Antagonist antibody for the treatment of cancer

ABSTRACT

Antibodies, humanized antibodies, resurfaced antibodies, antibody fragments, derivatized antibodies, and conjugates of same with cytotoxic agents, which specifically bind to, and inhibit A class of Eph receptors, antagonize the effects of growth factors on the growth and survival of tumor cells, and which have minimal agonistic activity or are preferrentially devoid of agonist activity. Said antibodies and fragments thereof may be used in the treatment of tumors that express elevated levels of A class of Eph receptors, such as breast cancer, colon cancer, lung cancer, ovarian carcinoma, synovial sarcoma and pancreatic cancer, and said derivatized antibodies may be used in the diagnosis and imaging of tumors that express elevated levels of A class of Eph receptors. Also provided are cytotoxic conjugates comprising a cell binding agent and a cytotoxic agent, therapeutic compositions comprising the conjugate, methods for using the conjugates in the inhibition of cell growth and the treatment of disease, and a kit comprising the cytotoxic conjugate are disclosed are all embodiments of the invention. In particular, the cell binding agent is a monoclonal antibody, and epitope-binding fragments thereof, that recognizes and binds the A class of Eph receptors.

FIELD OF THE INVENTION

The present invention provides novel murine anti-Eph monoclonalantibodies or fragments thereof, and humanized or resurfaced versionsthereof. More specifically, the invention relates to novel monoclonalantibodies or fragments thereof, and humanized or resurfaced versionsthereof, which interact with the EphA receptor family and act asantagonists. More particularly, the invention relates to anti-EphA2receptor antibodies that inhibit the cellular functions of the EphA2receptor. Still more particularly, the invention relates to anti-EphA2receptor antibodies that antagonize growth and survival of tumor cellsand which are devoid of agonist activity.

The present invention is further directed to cytotoxic conjugatescomprising a cell binding agent and a cytotoxic agent, therapeuticcompositions comprising the conjugate, methods for using the conjugatesin the inhibition of cell growth and the treatment of disease, and a kitcomprising the cytotoxic conjugate. In particular, the cell bindingagent is a monoclonal antibody, or epitope-binding fragment thereof, anda humanized or resurfaced version thereof that recognizes and binds theEphA family of receptors.

BACKGROUND OF THE INVENTION

Receptor tyrosine kinases play a diverse role in cell growth anddifferentiation during normal physiologic responses and in oncogenictransformation and tumor progression. Eph receptors are a unique familyof receptor tyrosine kinases (RTK), the largest in the genome,consisting of at least 16 receptors that interact with ninemembrane-bound ephrin ligands (Pasquale, E. B. et al., 2005, NatureReviews Mol. Cell Biol., 6: 462-475). They can be further divided intotwo groups, class A and B, based on the sequence homology and bindingaffinity (Pasquale, E. B. et al., 2005, Nature Reviews Mol. Cell Biol.,6: 462-475). Class A Eph receptors interact with multiple ligands of theephrin-A family, a group of glycosyl-phosphatidylinositol (GPI)-linkedmembrane proteins, while class B Eph receptors bind to ephrin-B ligands,a family of transmembrane proteins. Binding of Eph receptors to theirligands induces receptor clustering, activation of kinase activity, andsubsequent trans-phosphorylation of the cytoplasmic domains on tyrosineresidues, creating docking sites for a number of signaling proteins(Kullander, K. and Klein, R., 2002, Nature Reviews Mol. Cell Biol., 3:475-486; Noren, N. K. and Pasquale, E. B., 2004, Cell signal., 16:655-666).

Cancer is a disease characterized by uncontrolled proliferation,resulting from aberrant signal transduction. The most dangerous forms ofcancer are malignant cells which have the ability of these to spread,either by direct growth into adjacent tissue through invasion, or byimplantation into distant sites by metastasis. Metastatic cells haveacquired the ability to break away from the primary tumor, translocateto distant sites through the bloodstream or lymphatic system, andcolonize distant and foreign microenvironments.

It is now clear that the Eph molecules also have a role in diseasestates such as cancer. In particular, overexpression of the EphA2receptor has been reported in cancers of the ovary, breast, prostate,lung, colon, oesophagus, renal cell, cervix, and melanoma. EphA2 wassuggested to be a positive regulator of cell growth and survival inmalignant cells (Landen, C. N. et al., 2005, Expert. Opin. Ther.Targets, 9 (6): 1179-1187). A role for EphA2 in metastasis has also beendescribed, since EphA2 overexpression alone is sufficient to transformmammary epithelial cells into a malignant phenotype (Zelinski et al.,2001, Cancer Res., 61: 2301-2306), and increases spontaneous metastasisto distant sites (Landen, C. N. et al., 2005, Expert. Opin. Ther.Targets, 9 (6): 1179-1187). Furthermore, increasing evidence suggeststhat EphA2 is involved in tumor angiogenesis (Ogawa et al., 2000,Oncogene, 19: 6043-6052; Cheng et al. 2002, Mol. Cancer Res., 1: 2-11;Cheng et al., 2003, Neoplasia, 5 (5): 445-456; Dobrzanski et al., 2004,Cancer Res., 64: 910-919).

Phosphorylation of EphA2 has been shown to be linked to its abundance.Tyrosine phosphorylated EphA2 is rapidly internalised and fated fordegradation, whereas unphosphorylated EphA2 demonstrates reducedturnover and therefore accumulates at the cell surface. It is currentlythought that this kind of model might contribute to the high frequencyof EphA2 overexpression in cancer (Landen, C. N. et al., 2005, Expert.Opin. Ther. Targets, 9 (6): 1179-1187). However, reality may be morecomplex, since recent data seem to indicate a role for EphA2kinase-dependent and -independent functions in tumor progression (FangW. B., 2005, Oncogene, 24: 7859-7868).

Agonistic antibodies have been developped which promote EphA2 tyrosinephosphorylation and internalisation, ultimately resulting in inhibitionof tumor cell growth (Dodge-Zantek et al., 1999, Cell Growth & Differ.,10: 629-638; WO 01/12172, WO 03/094859, WO 2004/014292, WO 2004/101764,WO 2006/023403, WO 2006/047637, WO 2007/030642). These antibodies aredirected against the extracellular domain of EphA2. Since these agonistantibodies do not inhibit but rather stimulate EphA2 receptorphosphorylation and downstream signals, these antibodies might not beeffective for tumors which take advantage of the EphA2 kinase activity.On the other hand, the use of antagonistic agents, including antibodies,has been proposed (WO 2004/092343), but no actual antagonistic antibodywas disclosed therein. Moreover, such antibodies were proposed tostimulate, rather than inhibit, cell proliferation. Application WO2006/084226 discloses antibodies which neither increase nor decreaseEphA2 kinase activity but are capable of impeding tumor cellproliferation. However, there is no indication therein that theseantibodies prevent ephrinA1 binding to the receptor and inhibitephrinA1-induced EphA2 phosphorylation. Rather, they may affect tumorcell proliferation through a totally different mechanism, e.g. bypreventing receptor clustering following ephrinA1 binding. The skilledperson would thus not have concluded that these antibodies areantagonists, but, rather, that their mechanism of action is unclear.

Therefore, there is a need for new, antagonistic anti-EphA2 antibodies,which bind to the extracellular domains of EphA2 receptor, inhibit itsactivation by the ligand ephrin A1 and inhibit EphA2 kinase-dependendtumor cell growth. Such antagonistic antibodies should be useful for thetreatment of cancer.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide agents thatspecifically bind to class A Eph receptor family members, such as EphA2,and inhibit the cellular activity of the receptor by antagonizing thereceptor. Thus, the present invention includes antibodies or fragmentsthereof that recognize the EphA2 receptor, preferably human, andfunction as antagonists of said receptor.

The EphA2 receptor has a role in the development and the growth oftumors, and has also been involved in metastasis. In some embodiments,the antibodies of the invention are capable of inhibiting the growth ofa cancer cell. In some other embodiments, the antibodies of theinvention are capable of preventing the migration of metastatic cancercells. In preferred embodiments, the cancer cell is a cell of a cancerselected from the group consisting of a breast cancer, colon cancer,endometrial cancer, ovarian carcinoma, osteosarcoma, cervical cancer,prostate cancer, lung cancer, synovial carcinoma pancreatic cancer, asarcoma, a glioma, head and neck cancer, gastric cancer, liver cancer,and other carcinomas. In another embodiment, the antibodies of theinvention are capable of inhibiting angiogenesis.

Whereas the anti-EphA2 antibodies disclosed in the prior art were mostlyagonists (e.g. WO 03/094859, WO 2004/014292, WO 2004/101764, WO2006/023403, WO 2006/047637, WO 2007/030642), this invention encompassesantibodies recognizing said receptor wich have minimal agonisticactivity, or, preferentially, which are devoid of any agonist activitytowards the receptor. In a preferred embodiment, the antibodies of theinvention do not stimulate EphA2 tyrosine phosphorylation.

The antibodies of the invention are capable of inhibiting the binding ofa ligand, preferably ephrin A1, to the EphA2 receptor. In someembodiments, they are capable of inhibiting EphA2 tyrosinephosphorylation. In another embodiment, EphA2 tyrosine phosphorylationis inhibited by the antibodies of the invention even in the presence ofephrinA1. In some embodiments, antibodies of the invention can blockEphA2-mediated signaling; in particular, they are capable of inhibitingEphA2-dependent phosphorylation of Akt.

This invention also provides antibodies which bind the EphA2 receptorwith a K_(D) of 0.3×10⁻⁹ M or smaller.

Antibodies of the invention can be polyclonal or monoclonal.Epitope-binding fragments such as Fab, Fab′, F(ab′)₂, or Fv fragmentsare included within the scope of this invention. Preferred aremonoclonal anti-EphA2 antibodies. In a more preferred embodiment, thereare provided murine antibodies selected from 37.3D7; 37.1F5; 53.2H11;EphA2-N1; and EphA2-N2, which are fully characterized herein withrespect to the amino acid sequences of both their light and heavy chainvariable regions, the cDNA sequences of the genes for the light andheavy chain variable regions, the identification of their CDRs(complementarity-determining regions), the identification of theirsurface amino acids, and means for their expression in recombinant form.The hybridoma producing murine anti-EphA2 monoclonal antibodies 37.3D7,37.1F5, and 53.2H11, and EphA2-N1 and EphA2-N2 have been deposited underthe Budapest Treaty on Jun. 16, 2006 and on May 3, 2007, respectively,at the American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209, USA, under the accession numbers PTA-7660,PTA-7661, PTA-7662, PTA-8407, and PTA-8408, respectively.

The present invention includes the murine anti-EphA2 monoclonal antibodyselected from 37.3D7, 37.1F5, 53.2H11, EphA2-N1, and EphA2-N2, andresurfaced or humanized versions of the 37.3D7, 37.1F5, 53.2H11,EphA2-N1, and EphA2-N2 antibodies wherein surface-exposed residues ofthe variable region frameworks of the antibodies, or theirepitope-binding fragments, are replaced in both light and heavy chainsto more closely resemble known human antibody surfaces. The humanizedantibodies and epitope-binding fragments thereof of the presentinvention have improved properties in that they are less immunogenic (orcompletely non-immunogenic) than murine versions in human subjects towhich they are administered. Thus, the different versions of humanized37.3D7; 37.1F5; 53.2H11; EphA2-N1; and EphA2-N2 antibodies andepitope-binding fragments thereof of the present invention specificallyrecognize EphA2 receptor while not being immunogenic to a human.

The humanized versions of the 37.3D7, 37.1F5, 53.2H11, EphA2-N1, andEphA2-N2 antibodies of the present invention are fully characterizedherein with respect to their respective amino acid sequences of bothlight and heavy chain variable regions, the DNA sequences of the genesfor the light and heavy chain variable regions, the identification ofthe complementarity determining regions (CDRs), the identification oftheir variable region framework surface amino acid residues, anddisclosure of a means for their expression in recombinant form.

This invention also contemplates the use of conjugates between cytotoxicconjugates comprising (1) a cell binding agent that recognizes and bindsthe EphA receptor, such as, EphA2 receptor, and (2) a cytotoxic agent.In the cytotoxic conjugates, the cell binding agent has a high affinityfor the EphA receptor (e.g., EphA2 receptor) and the cytotoxic agent hasa high degree of cytotoxicity for cells expressing the EphA receptor,such that the cytotoxic conjugates of the present invention formeffective killing agents.

In a preferred embodiment, the cell binding agent is an anti-EphA2antibody (e.g., 37.3D7, 37.1F5, 53.2H11, EphA2-N1, or EphA2-N2) or anepitope-binding fragment thereof, more preferably a humanized anti-EphA2antibody (e.g., 37.3D7, 37.1F5, 53.2H11, EphA2-N1, or EphA2-N2) or anepitope-binding fragment thereof, wherein a cytotoxic agent iscovalently attached, directly or via a cleavable or non-cleavablelinker, to the antibody or epitope-binding fragment thereof. In morepreferred embodiments, the cell binding agent is the humanized 37.3D7;37.1F5; 53.2H11; EphA2-N1; and EphA2-N2 antibodies or an epitope-bindingfragment thereof, and the cytotoxic agent is a taxol, a maytansinoid, atomaymycin derivative, a leptomycin derivative, CC-1065 or a CC-1065analog.

In preferred embodiments of the invention, the cell binding agent is thehumanized anti-EphA2 antibody 37.3D7, 37.1F5, 53.2H11, EphA2-N1, orEphA2-N2 and the cytotoxic agent is a maytansine compound, such as DM1or DM4.

The present invention also encompasses the use of fragments ofanti-EphA2 antibodies which retain the ability to bind the EphA2receptor. In another aspect of the invention, the use of functionalequivalents of anti-EphA2 antibodies is contemplated.

The present invention also includes a method for inhibiting the growthof a cell expressing the EphA2 receptor. In preferred embodiments, themethod for inhibiting the growth of the cell expressing the EphA2receptor takes place in vivo and results in the death of the cell,although in vitro and ex vivo applications are also included.

The present invention also provides a therapeutic composition comprisingan anti-EphA2 antibody or an anti-EphA2 antibody-cytotoxic agentconjugate, and a pharmaceutically acceptable carrier or excipients. Insome embodiments, the therapeutic composition comprises a secondtherapeutic agent. This second therapeutic agent can be chosen from thegroup comprising the antagonists of fibroblast-growth factor (FGF),hepatocyte growth factor (HGF), tissue factor (TF), protein C, proteinS, platelet-derived growth factor (PDGF), or HER2 receptor.

The present invention further includes a method of treating a subjecthaving cancer using the therapeutic composition. In some embodiments,the cancer is a metastatic cancer. In particular, the cancer cell is acell of a cancer selected from the group consisting of breast cancer,colon cancer, endometrial cancer, ovarian carcinoma, osteosarcoma,cervical cancer, prostate cancer, lung cancer, synovial carcinomapancreatic cancer, a sarcoma, a glioma, head and neck cancer, gastriccancer, liver cancer, and other carcinomas. In preferred embodiments,the cytotoxic conjugate comprises an anti-EphA2 antibody and a cytotoxicagent. In more preferred embodiments, the cytotoxic conjugate comprisesa humanized 37.3D7, 37.1F5, 53.2H11, EphA2-N1, and EphA2-N2 antibody-DM1conjugate, humanized 37.3D7, 37.1F5, 53.2H11, EphA2-N1, and EphA2-N2antibody-DM4, a humanized 37.3D7, 37.1F5, 53.2H11, EphA2-N1, andEphA2-N2 antibody-taxane conjugate, or a humanized 37.3D7, 37.1F5,53.2H11, EphA2-N1, and EphA2-N2 antibody-tomaymycin derivativeconjugate, and the conjugate is administered along with apharmaceutically acceptable carrier or excipients.

In another aspect of the invention, anti-EphA2 antibodies are used todetect the EphA2 protein in a biological sample. In a preferredembodiment, said antibodies are used to determine EphA2 levels in atumor tissue.

The present invention also includes a kit comprising an anti-EphA2antibody or an anti-EphA2 antibody-cytotoxic agent conjugate andinstructions for use. In preferred embodiments, the anti-EphA2antibodies are the humanized 37.3D7, 37.1F5, 53.2H11, EphA2-N1, andEphA2-N2 antibodies, the cytotoxic agent is a maytansine compound, suchas DM1 or DM4, a taxane, a leptomycin derivative, or a tomaymycinderivative, and the instructions are for using the conjugates in thetreatment of a subject having cancer. The kit may also includecomponents necessary for the preparation of a pharmaceuticallyacceptable formulation, such as a diluent if the conjugate is in alyophilized state or concentrated form, and for the administration ofthe formulation.

Unless otherwise stated, all references and patents cited herein areincorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C show the analysis of the specific binding of anti-EphA2antibodies to cells overexpressing human EphA2 (300-19/hu-EphA2 cells)by FACS analysis. FIG. 1A shows the data for the 37.3D7 antibody, FIG.1B for the 37.1F5 antibody, and FIG. 1C for the 53.2H11 antibody,respectively.

FIG. 2 shows the specific binding of purified 37.3D7 antibody to BxPC3human pancreatic cancer cells, MDA-MB-231 human breast cancer cells, andHT-29 human colon cancer cells. Histograms of FACS analysis are shown.

FIGS. 3A-3C show binding curves for the antibodies 37.3D7 (FIG. 3A),37.1F5 (FIG. 3B), and 53.2H11 (FIG. 3C) established with human EphA2overexpressing murine 300-19 cells (300-19/hu-EphA2).

FIG. 4 shows the specific binding of purified 37.3D7 and 53.2H11antibodies to cells overexpressing EphA2. Histograms of FACS analysisare shown. FIG. 4A shows the data for cells overexpressing murine EphA2(300-19/mu-EphA2) and FIG. 4B shows the data for cells overexpressingrat EphA2 (300-19/rat-EphA2).

FIG. 5A shows the specific binding of purified 37.3D7, 37.1F5, and53.2H11 antibodies to VERO monkey kidney epithelial cells. Histograms ofFACS analysis are shown.

FIG. 5B shows the binding curves for the antibodies 37.3D7, 37.1F5, and53.2H11 established with VERO monkey kidney epithelial cells.

FIG. 6 shows the inhibition of the binding of biotinylated ephrinA1 tomammary MDA-MB-231 human breast cancer cells by 37.3D7, 37.1F5, and53.2H11 antibodies.

FIG. 7A shows the inhibition of ephrinA1-stimulatedEphA2-phosphorylation in mammary MDA-MB-231 cells by 37.3D7 and 37.1F5antibodies.

FIG. 7B shows the inhibition of ephrinA1-stimulatedEphA2-phosphorylation in mammary MDA-MB-231 cells by 37.3D7 and 53.2H11antibodies.

FIG. 7C shows the inhibition of ephrinA1-stimulated Akt phosphorylationin pancreatic CFPAC-1 cells by 37.3D7 and 37.1F5 antibodies.

FIGS. 8A and B show the stimulation of EphA2-phosphorylation by ephrinA1and the absence of stimulation of EphA2-phosphorylation by theantibodies 37.3D7, 37.1F5, and 53.2H11 in mammary MDA-MB-231 cells.

FIGS. 9A-9D show the inhibition of serum-stimulated growth and survivalof colon HT-29 cells (9A), colon LoVo cells (9B), pancreatic CFPAC-1cells (9C) and melanoma UACC-257 cells (9D) by 37.3D7 and 53.2H11antibodies.

FIG. 10A shows the dose-dependent inhibition of serum-stimulated growthof pancreatic BxPC3 cells by 37.3D7 antibody.

FIG. 10B shows the dose-dependent inhibition of serum-stimulated growthof colon LoVo cells by the 53.2H11 antibody.

FIG. 10C shows the dose-dependent inhibition of EGF-stimulated growth ofcolon LoVo cells by 53.2H11 antibody.

FIG. 11A shows the binding curve of 37.3D7 antibody to HUVEC cells.

FIG. 11B shows the binding curve of 37.1F5 antibody to HUVEC cells.

FIG. 12 shows the inhibition of VEGF-stimulated HUVEC cell growth andsurvival by 37.3D7 antibody.

FIG. 13 shows the inhibition of VEGF-induced Akt phosphorylation by37.3D7 antibody in HUVEC cells.

FIG. 14 shows the effect of the treatment with 37.3D7 antibody on thegrowth of HT-29 colon cancer xenograft in mice. The effect is comparedwith that of an anti-EGFR antibody and a non-binding control IgG1antibody.

FIG. 15A shows the inhibition of the growth of PC3 prostate tumor cellsby hu37.3D7-SPDB-DM4.

FIG. 15B shows the inhibition of the growth of PC3 prostate tumor cellsby hu53.2H11-SPDB-DM4.

FIG. 16A shows the effect of the treatment with hu37.3D7-SPDB-DM4 on thegrowth of MDA-MB-231 breast tumor xenograft in mice.

FIG. 16B shows the effect of the treatment with hu53.2H11-SPDB-DM4 onthe growth of MDA-MB-231 breast tumor xenograft in mice.

DETAILED DESCRIPTION OF THE INVENTION

New agents capable to specifically bind EphA receptors and antagonizesaid receptors are herein provided. In particular, the present inventorshave discovered novel antibodies that specifically bind to EphAreceptors on the cell surface. While previously known antibodies whichspecifically bind the EphA receptor also activate it even in the absenceof its ligands, the antibodies or fragments of the present invention arepreferentially devoid of any agonist activity. On the other hand, theyhave the unique ability to inhibit the cellular functions of thereceptor even in the presence of its ligands, a characteristic which istotally absent from the previously known EphA2-binding antibodies.Furthermore, the antagonistic antibodies and antibody fragments of thepresent invention inhibit the growth and/or the migration of human tumorcells, and/or angiogenesis, three properties totally unanticipated inview of the prior art (Landen, C. N. et al., 2005, Expert. Opin. Ther.Targets, 9 (6): 1179-1187; WO 01/12172; WO 2004/014292; WO 2004/092343).

As used herein, the term “Eph receptor” refers to a tyrosine kinasebelonging to the Eph receptors family (reviewed in Pasquale, E. B. etal., 2005, Nature Reviews Mol. Cell Biol., 6, 462-475). “Class A Ephreceptor family” or “EphA receptors” as used herein preferentiallyinteract with glycosylphosphatidylinositol (GPI)-linked ligands (of theEphrin-A subclass, which presently comprises five ligands). SpecificEphA receptors include: EphA1 (also called Eph and Esk); EphA2 (alsocalled Eck, mEck, Myk2, Sek2); EphA3 (also termed Hek, Mek4, Tyro4 andCek4); EphA4 (also known as Hek8, Sek1, Tyrol, and Cek8); EphA5 (alsocalled Hek7, Bsk, Ehk1, Rek7 and Cek7); EphA6 (also called mEhk2 andEhk2); EphA7 (otherwise named Hek11, Mdk1, Ebk, Ehk3); and EphA8 (alsotermed Eek and mEek) and naturally occurring variants thereof. Thepreferred Eph receptor herein is the “EphA2 receptor”, comprising, forexample, an amino sequence as in Genbank accession Nos NM_(—)004431(human EphA2), NM_(—)010139 (murine EphA2), or NXM_(—)345596 (ratEphA2). The term “Eph ligand” as used herein refers to a protein thatbinds to, and optionally activates (e.g. stimulates theautophosphorylation of), an Eph receptor. A preferred Eph ligand hereinis “ephrinA1”, which binds to the EphA2 receptor and comprises, forexample, an amino sequence as in Genbank accession NM_(—)004428 (humanephrinA1).

The term “antagonist” as used herein refers to a molecule which iscapable of inhibiting one or more of the biological activities of atarget molecule, such as an EphA receptor. Antagonists may act byinterfering with the binding of a receptor to a ligand and vice versa,by decreasing EphA2 phosphorylation, and/or by incapacitating or killingcells which have been activated by a ligand. The antagonist maycompletely block receptor-ligand interactions or may substantiallyreduce such interactions. All such points of intervention by anantagonist shall be considered equivalent for purposes of thisinvention. Thus, included within the scope of the invention areantagonists (e.g. neutralizing antibodies) that bind to EphA receptor,Eph ligand or a complex of an Eph receptor and Eph ligand; amino acidsequence variants or derivatives of an EphA receptor or EphA ligandwhich antagonize the interaction between an EphA receptor and EphAligand; soluble EphA receptor or soluble EphA ligand, optionally fusedto a heterologous molecule such as an immunoglobulin region (e.g. animmunoadhesin); a complex comprising an EphA receptor in associationwith EphA ligand; synthetic or native sequence peptides which bind toEphA receptor or EphA ligand.

The term “agonist” as used herein refers to any compound, including aprotein, a polypeptide, a peptide, an antibody, an antibody fragment, aconjugate, a large molecule, a small molecule, capable of activating oneor more of the biological activities of the target molecule. EphAagonists act by stimulating phosphorylation of the protein, therebytriggering degradation of said protein.

Thus in a preferred embodiment the present invention provides, amongother features, anti-EphA monoclonal antibodies, anti-EphA humanizedantibodies, and fragments of the anti-EphA antibodies. Each of theantibodies and antibody fragments of the present invention is designedto specifically recognize and bind the EphA2 receptor, and acts as anEphA2 receptor antagonist. Moreover, the antagonistic antibodies andantibody fragments of the invention have the unique properties of beingable to inhibit the growth of human tumor cells, and/or the migration ofmetastatic cancer cells, and/or angiogenesis.

A preferred EphA receptor bound by the antagonistic antibodies andantibody fragments of the invention is the EphA2 receptor. Human EphA2is a preferred EphA2 receptor.

The EphA2 receptor belongs to a family of receptor whose cytoplasmictail phosphorylation is increased after ligand binding to interact witha variety of adapter and signalling proteins, leading to the activationof different downstream cellular signalling pathways (Kullander, K. andKlein, R., 2002, Nature Reviews Mol. Cell Biol., 3: 475-486; Noren, N.K. and Pasquale, E. B., 2004, Cell signal., 16: 655-666). As usedherein, the term “EphA2-mediated signaling” refers to all the cellularevents which occur in response to ligand binding by EphA2. Whereasantibodies disclosed in the prior art agonize the EphA2 receptor, and,in particular, increase the tyrosine phosphorylation of the EphA2protein, the antibodies and antibody fragments of the invention arepreferentially devoid of any such agonistic properties. In particular,they are unable to stimulate EphA2 phoshorylation by themselves.

On the other hand, this invention provides the first actual antagonisticanti-EphA2 antibodies. In one embodiment, the antibodies and antibodyfragments of the invention can inhibit the binding of a ligand to anEphA receptor. In a preferred embodiment, the binding of ephrinA1 toEphA2 is prevented by the antibodies and fragments thereof provided bythis invention. Remarkably, in another embodiment, the antibodies andantibody fragments of the invention are capable of inhibiting tyrosinephosphorylation of the EphA2 receptor, even in the presence of ephrinA1.Moreover, said antibodies and fragments thereof are capable ofinhibiting EphA2-mediated signaling. In particular, AktephrinA1-dependent phosphorylation can be prevented by the antibodiesand antibody fragments of the invention.

Antibodies

The term “antibody” is used herein in the broadest sense andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies) of any isotype such as IgG, IgM, IgA, IgD, andIgE, polyclonal antibodies, multispecific antibodies, chimericantibodies, and antibody fragments. An antibody reactive with a specificantigen can be generated by recombinant methods such as selection oflibraries of recombinant antibodies in phage or similar vectors, or byimmunizing an animal with the antigen or an antigen-encoding nucleicacid.

A typical antibody is comprised of two identical heavy chains and twoidentical light chains that are joined by disulfide bonds. Each heavyand light chain contains a constant region and a variable region. Eachvariable region contains three segments called“complementarity-determining regions” (“CDRs”) or “hypervariableregions”, which are primarily responsible for binding an epitope of anantigen. They are usually referred to as CDR1, CDR2, and CDR3, numberedsequentially from the N-terminus. The more highly conserved portions ofthe variable regions are called the “framework regions”.

As used herein, “V_(H)” or “VH” refers to the variable region of animmunoglobulin heavy chain of an antibody, including the heavy chain ofan Fv, scFv, dsFv, Fab, Fab′, or F(ab′)2 fragment. Reference to “V_(L)”or “VL” refers to the variable region of the immunoglobulin light chainof an antibody, including the light chain of an Fv, scFv, dsFv, Fab,Fab′, or F(ab′)2 fragment.

A “polyclonal antibody” is an antibody which was produced among or inthe presence of one or more other, non-identical antibodies. In general,polyclonal antibodies are produced from a B-lymphocyte in the presenceof several other B-lymphocytes producing non-identical antibodies.Usually, polyclonal antibodies are obtained directly from an immunizedanimal.

A “monoclonal antibody”, as used herein, is an antibody obtained from apopulation of substantially homogeneous antibodies, i.e. the antibodiesforming this population are essentially identical except for possiblenaturally occurring mutations which might be present in minor amounts.These antibodies are directed against a single epitope and are thereforehighly specific.

An “epitope” is the site on the antigen to which an antibody binds. Itcan be formed by contiguous residues or by non-contiguous residuesbrought into close proximity by the folding of an antigenic protein.Epitopes formed by contiguous amino acids are typically retained onexposure to denaturing solvents, whereas epitopes formed bynon-contiguous amino acids are typically lost under said exposure.

As used herein, the term “K_(D)” refers to the dissociation constant ofa particular antibody/antigen interaction.

The present invention proceeds from murine anti-EphA2 antibodies, herein37.3D7; 37.1F5; 53.2H11; EphA2-N1; and EphA2-N2 which are fullycharacterized with respect to the amino acid sequences of both light andheavy chains, the identification of the CDRs, the identification ofsurface amino acids, and means for their expression in recombinant form.The primary amino acid and DNA sequences of antibodies 37.3D7; 37.1F5;53.2H11; EphA2-N1; and EphA2-N2 light and heavy chains, and of humanizedversions, are disclosed herein.

Antibodies 37.3D7, 37.1F5, 53.2H11, EphA2-N1, and EphA2-N2 are producedby hybridomas respectively designated 37.3D7, 37.1F5, 53.2H11, EphA2-N1,and EphA2-N2, and deposited under the Budapest Treaty on Jun. 16, 2006and May 3, 2007, respectively, at the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110-2209, USA, under theaccession numbers PTA-7660, PTA-7661 PTA-7662, PTA-8407 and PTA-8408,respectively.

The scope of the present invention is not limited to antibodies andfragments comprising these sequences. Instead, all antibodies andfragments that specifically bind to EphA2 receptor and antagonize thebiological activity of the receptor, but which are devoid of agonistactivity, fall within the scope of the present invention. Thus,antibodies and antibody fragments may differ from antibody 37.3D7;37.1F5; 53.2H11; EphA2-N1; and EphA2-N2 or the humanized derivatives inthe amino acid sequences of their scaffold, CDRs, light chain and heavychain, and still fall within the scope of the present invention.

In one embodiment, this invention provides antibodies or epitope-bindingfragment thereof comprising one or more CDRs having an amino acidsequence selected from the group consisting of SEQ ID NOS: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, and 72.

In a preferred embodiment, the antibodies of the invention comprise atleast one heavy chain and at least one light chain, and said heavy chaincomprises three sequential CDRs having amino acid sequences selectedfrom the group consisting of SEQ ID NOS: 1, 2, 3, 7, 8, 9, 13, 14, 15,61, 62, 63, 67, 68, and 69, and said light chain comprises threesequential CDRs having amino acid sequences selected from the groupconsisting of SEQ ID NOS: 4, 5, 6, 10, 11, 12, 16, 17, 18, 64, 65, 66,70, 71, and 72.

In a more preferred embodiment, the antibodies of the invention comprisethree CDRS having amino acid sequences selected from the group of SEQ IDNOS: 1, 2, 3, 4, 5, and 6. In a further more preferred embodiment, thereis provided a 37.3D7 antibody, which comprises at least one heavy chainand at least one light chain, and said heavy chain comprises threesequential CDRs having amino acid sequences consisting of SEQ ID NOS: 1,2, and 3, and said light chain comprises three sequential CDRs havingamino acid sequences consisting of SEQ ID NOS: 4, 5, and 6.

In another more preferred embodiment, the antibodies of the inventioncomprise three CDRS having amino acid sequences selected from the groupof SEQ ID NOS: 7, 8, 9, 10, 11, and 12. In further more preferredembodiment, there is provided a 37.1F5 antibody, which comprises atleast one heavy chain and at least one light chain, and said heavy chaincomprises three sequential CDRs having amino acid sequences consistingof SEQ ID NOS: 7, 8, and 9, and said light chain comprises threesequential CDRs having amino acid sequences consisting of SEQ ID NOS:10, 11, and 12.

In another more preferred embodiment, the antibodies of the inventioncomprise three CDRS having amino acid sequences selected from the groupof SEQ ID NOS: 13, 14, 15, 16, 17, and 18. In a further more preferredembodiment, there is provided a 53.2H11, which comprises at least oneheavy chain and at least one light chain, and said heavy chain comprisesthree sequential CDRs having amino acid sequences consisting of SEQ IDNOS: 13, 14, and 15, and said light chain comprises three sequentialCDRs having amino acid sequences consisting of SEQ ID NOS: 16, 17, and18.

In another more preferred embodiment, the antibodies of the inventioncomprise three CDRS having amino acid sequences selected from the groupof SEQ ID NOS: 61, 62, 63, 64, 65, and 66. In a further more preferredembodiment, there is provided a EphA2-N1 antibody, which comprises atleast one heavy chain and at least one light chain, and said heavy chaincomprises three sequential CDRs having amino acid sequences consistingof SEQ ID NOS: 61, 62, and 63, and said light chain comprises threesequential CDRs having amino acid sequences consisting of SEQ ID NOS:64, 65, and 66.

In another more preferred embodiment, the antibodies of the inventioncomprise three CDRS having amino acid sequences selected from the groupof SEQ ID NOS: 67, 68, 69, 70, 71, and 72. In a further more preferredembodiment, there is provided a EphA2-N2 antibody, which comprises atleast one heavy chain and at least one light chain, and said heavy chaincomprises three sequential CDRs having amino acid sequences consistingof SEQ ID NOS: 67, 68, and 69, and said light chain comprises threesequential CDRs having amino acid sequences consisting of SEQ ID NOS:70, 71, and 72.

In another embodiment, the antibodies of the invention comprises a V_(H)having an amino acid sequence selected from the group consisting of SEQID NOS: 20, 22, 24, 74 and 76. In a preferred embodiment, there isprovided a 37.3D7 antibody comprising a V_(H) having an amino acidsequence consisting of SEQ ID NO 20. In another preferred embodiment,there is provided a 37.1F5 antibody comprising a V_(H) having an aminoacid sequence consisting of SEQ ID NO 22. In another preferredembodiment, there is provided a 53.2H11 antibody comprising a V_(H)having an amino acid sequence consisting of SEQ ID NO 24. In anotherpreferred embodiment, there is provided a EphA2-N1 antibody comprising aV_(H) having an amino acid sequence consisting of SEQ ID NO 74. Inanother preferred embodiment, there is provided a EphA2-N2 antibodycomprising a V_(H) having an amino acid sequence consisting of SEQ ID NO76.

In another preferred embodiment, the antibodies of the inventioncomprise a V_(L) having an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 26, 28, 30, 78 and 80. In a preferredembodiment, there is provided a 37.3D7 antibody comprising a V_(L)having an amino acid sequence consisting of SEQ ID NO 26. In anotherpreferred embodiment, there is provided a 37.1F5 antibody comprising aV_(L) having an amino acid sequence consisting of SEQ ID NO 28. Inanother preferred embodiment, there is provided a 53.2H11 antibodycomprising a V_(L) having an amino acid sequence consisting of SEQ ID NO30. In another preferred embodiment, there is provided a EphA2-N1antibody comprising a V_(L) having an amino acid sequence consisting ofSEQ ID NO 78. In another preferred embodiment, there is provided aEphA2-N2 antibody comprising a V_(L) having an amino acid sequenceconsisting of SEQ ID NO 80.

Humanized or Resurfaced 37.3D7, 37.1F5; 53.2H11; EphA2-N1; and EphA2-N2Antibodies

As used herein, the term “humanized antibody” refers to a chimericantibody which contain minimal sequence derived from non-humanimmunoglobulin. A “chimeric antibody”, as used herein, is an antibody inwhich the constant region, or a portion thereof, is altered, replaced,or exchanged, so that the variable region is linked to a constant regionof a different species, or belonging to another antibody class orsubclass. “Chimeric antibody” also refers to to an antibody in which thevariable region, or a portion thereof, is altered, replaced, orexchanged, so that the constant region is linked to a variable region ofa different species, or belonging to another antibody class or subclass.

The goal of humanization is a reduction in the immunogenicity of axenogenic antibody, such as a murine antibody, for introduction into ahuman, while maintaining the full antigen binding affinity andspecificity of the antibody. Humanized antibodies, or antibodies adaptedfor non-rejection by other mammals, may be produced using severaltechnologies such as resurfacing and CDR grafting. As used herein, theresurfacing technology uses a combination of molecular modeling,statistical analysis and mutagenesis to alter the non-CDR surfaces ofantibody variable regions to resemble the surfaces of known antibodiesof the target host.

Strategies and methods for the resurfacing of antibodies, and othermethods for reducing immunogenicity of antibodies within a differenthost, are disclosed in U.S. Pat. No. 5,639,641, which is herebyincorporated in its entirety by reference. Briefly, in a preferredmethod, (1) position alignments of a pool of antibody heavy and lightchain variable regions is generated to give a set of heavy and lightchain variable region framework surface exposed positions wherein thealignment positions for all variable regions are at least about 98%identical; (2) a set of heavy and light chain variable region frameworksurface exposed amino acid residues is defined for a rodent antibody (orfragment thereof); (3) a set of heavy and light chain variable regionframework surface exposed amino acid residues that is most closelyidentical to the set of rodent surface exposed amino acid residues isidentified; (4) the set of heavy and light chain variable regionframework surface exposed amino acid residues defined in step (2) issubstituted with the set of heavy and light chain variable regionframework surface exposed amino acid residues identified in step (3),except for those amino acid residues that are within 5 Å of any atom ofany residue of the complementarity-determining regions of the rodentantibody; and (5) the humanized rodent antibody having bindingspecificity is produced.

Antibodies can be humanized using a variety of other techniquesincluding CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos.5,530,101; and 5,585,089), veneering or resurfacing (EP 0 592 106; EP 0519 596; Padlan E. A., 1991, Molecular Immunology 28(4/5): 489-498;Studnicka G. M. et al., 1994, Protein Engineering 7(6): 805-814; RoguskaM. A. et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91:969-973), andchain shuffling (U.S. Pat. No. 5,565,332). Human antibodies can be madeby a variety of methods known in the art including phage displaymethods. See also U.S. Pat. Nos. 4,444,887, 4,716,111, 5,545,806, and5,814,318; and international patent application publication numbers WO98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741 (said references incorporated by reference intheir entireties).

The present invention provides humanized antibodies or fragmentsthereof, which recognizes EphA2 receptor and acts as antagonists. Inanother embodiment, the humanized antibodies or epitope-bindingfragments thereof have the additional ability to inhibit growth of acancer cell expressing the EphA2 receptor. In a further embodiment, thehumanized antibody or epitope-binding thereof have the additionalability to inhibit the migration of a metastatic cancer cell expressingthe EphA2 receptor.

A preferred embodiment of such a humanized antibody is a humanized37.3D7, 37.1F5; 53.2H11; EphA2-N1 or EphA2-N2 antibody, or anepitope-binding fragment thereof.

In more preferred embodiments, there are provided resurfaced orhumanized versions of the 37.3D7, 37.1F5; 53.2H11; EphA2-N1 and EphA2-N2antibodies wherein surface-exposed residues of the antibody or itsfragments are replaced in both light and heavy chains to more closelyresemble known human antibody surfaces. The humanized 37.3D7, 37.1F5;53.2H11; EphA2-N1 and EphA2-N2 antibodies or epitope-binding fragmentsthereof of the present invention have improved properties. For example,humanized 37.3D7, 37.1F5; and 53.2H11 antibodies or epitope-bindingfragments thereof specifically recognize EphA2 receptor. Morepreferably, the humanized 37.3D7, 37.1F5, 53.2H11, EphA2-N1, andEphA2-N2 antibodies or epitope-binding fragments thereof have theadditional ability to inhibit growth of a cell expressing the EphA2receptor.

The humanized versions of the 37.3D7, 37.1F5, 53.2H11, EphA2-N1, andEphA2-N2 antibodies are also fully characterized herein with respect totheir respective amino acid sequences of both light and heavy chainvariable regions, the DNA sequences of the genes for the light and heavychain variable regions, the identification of the CDRs, theidentification of their surface amino acids, and disclosure of a meansfor their expression in recombinant form. However, the scope of thepresent invention is not limited to antibodies and fragments comprisingthese sequences. Instead, all antibodies and fragments that specificallybind to EphA2 receptor are included in the present invention.Preferably, the antibodies and fragments that specifically bind to EphA2receptor antagonize the biological activity of the receptor. Morepreferably, such antibodies further are substantially devoid of agonistactivity. Thus, antibodies and epitope-binding antibody fragments of thepresent invention may differ from the 37.3D7, 37.1F5, 53.2H11, EphA2-N1or EphA2-N2 antibody or the humanized derivatives thereof, in the aminoacid sequences of their scaffold, CDRs, and/or light chain and heavychain, and still fall within the scope of the present invention.

The CDRs of the 37.3D7, 37.1F5, 53.2H11, EphA2-N1 or EphA2-N2 antibodiesare identified by modeling and their molecular structures have beenpredicted. Again, while the CDRs are important for epitope recognition,they are not essential to the antibodies and fragments of the invention.Accordingly, antibodies and fragments are provided that have improvedproperties produced by, for example, affinity maturation of an antibodyof the present invention.

The mouse light chain IgVκ and Jκ germline genes and heavy chain IgVhand Jh germline genes from which 37.3D7, 37.1F5, 53.2H11, EphA2-N1, andEphA2-N2 were likely derived have been identified, as disclosed in theexperimental Examples section. Such germline gene sequences are usefulto identify somatic mutations in the antibodies, including in the CDRs.

The sequences of the heavy chain and light chain variable regions of the37.3D7, 37.1F5, 53.2H11, EphA2-N1, and EphA2-N2 antibodies, and thesequences of their CDRs were not previously known and are set forth inthis application. Such information can be used to produce humanizedversions of the 37.3D7, 37.1F5, 53.2H11, EphA2-N1, and EphA2-N2antibodies. These humanized anti-EphA antibodies or their derivativesmay also be used as the cell binding agent of the present invention.

Thus, in one embodiment, this invention provides humanized antibodies orepitope-binding fragment thereof comprising one or more CDRs having anamino acid sequence selected from the group consisting of SEQ ID NOS: 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, and 72. In a preferred embodiment, thehumanized antibodies of the invention comprise at least one heavy chainand at least one light chain, and said heavy chain comprises threesequential CDRs having amino acid sequences selected from the groupconsisting of SEQ ID NOS: 1, 2, 3, 7, 8, 9, 13, 14, 15, 61, 62, 63, 67,68, and 69, and said light chain comprises three sequential CDRs havingamino acid sequences selected from the group consisting of SEQ ID NOS:4, 5, 6, 10, 11, 12, 16, 17, 18, 64, 65, 66, 70, 71, and 72. In afurther preferred embodiment, the humanized antibodies of the inventioncomprise at least one heavy chain and at least one light chain, whereinsaid heavy chain comprises three sequential CDRs having amino acidsequences represented by SEQ ID NOS: 1, 2, and 3, and wherein said lightchain comprises three sequential CDRs having amino acid sequencesrepresented by SEQ ID NOS: 4, 5, and 6. In another further preferredembodiment, the humanized antibodies of the invention comprise at leastone heavy chain and at least one light chain, wherein said heavy chaincomprises three sequential CDRs having amino acid sequences representedby SEQ ID NOS: 7, 8, and 9, and wherein said light chain comprises threesequential CDRs having amino acid sequences represented by SEQ ID NOS:10, 11, and 12. In another further preferred embodiment, the humanizedantibodies of the invention comprise at least one heavy chain and atleast one light chain, wherein said heavy chain comprises threesequential CDRs having amino acid sequences represented by SEQ ID NOS:13, 14, and 15, and wherein said light chain comprises three sequentialCDRs having amino acid sequences represented by SEQ ID NOS: 16, 17, and18. In another more preferred embodiment, the humanized antibodies ofthe invention comprise at least one heavy chain and at least one lightchain, wherein said heavy chain comprises three sequential CDRs havingamino acid sequences consisting of SEQ ID NOS: 61, 62, and 63, andwherein said light chain comprises three sequential CDRs having aminoacid sequences consisting of SEQ ID NOS: 64, 65, and 66. In another morepreferred embodiment, the humanized antibodies of the invention compriseat least one heavy chain and at least one light chain, wherein saidheavy chain comprises three sequential CDRs having amino acid sequencesconsisting of SEQ ID NOS: 67, 68, and 69, and wherein said light chaincomprises three sequential CDRs having amino acid sequences consistingof SEQ ID NOS: 70, 71, and 72.

In one embodiment, this invention provides humanized antibodies orfragments thereof which comprise a V_(H) having an amino acid sequencechosen from the group consisting of SEQ ID NOS: 32, 34, 36, 37, 38, 40,42, 43, and 45. In a preferred embodiment, a humanized 37.1 D7 antibodyis provided which comprises a V_(H) having an amino acid sequence chosenfrom the group consisting of SEQ ID NOS: 32, 34, and 36. In anotherpreferred embodiment, a humanized 37.1F5 antibody is provided whichcomprises a V_(H) having an amino acid sequence chosen from the groupconsisting of SEQ ID NOS: 37 and 38. In another preferred embodiment, ahumanized 53.2H11 antibody is provided which comprises a V_(H) having anamino acid sequence chosen from the group consisting of SEQ ID NOS: 40,42, 43, and 45.

In another embodiment, this invention provides humanized antibodies orfragments thereof which comprise a V_(L) having an amino acid sequencechosen from the group consisting of SEQ ID NOS: 47, 48, 49, 50, and 52.In a preferred embodiment, a humanized 37.1 D7 antibody is providedwhich comprises a V_(L) having an amino acid sequence consisting of SEQID NO 47. In another preferred embodiment, a humanized 37.1F5 antibodyis provided which comprises a V_(L) having an amino acid sequence chosenfrom the group consisting of SEQ ID NOS: 48, 49, and 50. In anotherpreferred embodiment, a humanized 53.2H11 antibody is provided whichcomprises a V_(L) having an amino acid sequence consisting of SEQ ID NO52.

The humanized 37.3D7 antibodies and epitope-binding fragments thereof ofthe present invention can also include substitution in light and/orheavy chain amino acid residues at one or more positions defined by thegrey residues in Table 1A and 1B which represent the murine surfaceframework residues that have been changed from the original murineresidue to the corresponding framework surface residue in the humanantibody, 28E4. The starred (*) residues in Table 1B correspond to themurine back mutations in the humanized 37.3D7 heavy chain variants (SEQID NO: 34 and SEQ ID NO:36). The residues for back mutations areproximal to CDR's and were chosen as described in U.S. Pat. No.5,639,641 or in analogy to the selection of residues that had inprevious humanization efforts resulted in a decrease in antigen bindingaffinity (Roguska et al., 1996, Protein Eng.; 9(10): 895-904; U.S.patent application publications 2003/0235582 and 2005/0118183).

Likewise, the humanized 37.1F5; 53.2H11; EphA2-N1; and EphA2-N2antibodies and epitope-binding fragments thereof of the presentinvention can also include substitution in light and/or heavy chainamino acid residues.

Polynucleotides, Vectors, and Host Cells

Nucleic acids encoding anti-EphA2 antibodies of the invention areprovided. In one embodiment, the nucleic acid molecule encodes a heavyand/or a light chain of an anti-EphA2 immunoglobulin. In a preferredembodiment, a single nucleic acid encodes a heavy chain of an anti-EphA2immunoglobulin and another nucleic acid molecule encodes the light chainof an anti-EphA2 immunoglobulin.

In another aspect of this invention, there are provided polynucleotidesencoding polypeptides having an amino acid sequence selected from thegroup of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 37, 38, 40, 42, 43, 45,47, 48, 49, 50, 52, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 74,76, 78 and 80. In a preferred embodiment, the polynucleotide of theinvention is selected from the group consisting of SEQ ID NOs: 19, 21,23, 25, 27, 29, 31, 33, 35, 39, 41, 44, 46, 51, 73, 75, 77, and 79. Theinvention is not limited to said polynucleotides per se but alsoincludes all polynucleotides displaying at least 80% identity with saidpolynucleotides.

The invention provides vectors comprising the polynucleotides of theinvention. In one embodiment, the vector contains a polynucleotideencoding a heavy chain of an anti-EphA2 immunoglobulin. In anotherembodiment, said polynucleotide encodes the light chain of an anti-EphA2immunoglobulin. The invention also provides vectors comprisingpolynucleotide molecules encoding fusion proteins, modified antibodies,antibody fragments, and probes thereof.

In order to express the heavy and/or light chain of the anti-EphA2antibodies of the invention, the polynucleotides encoding said heavyand/or light chains are inserted into expression vectors such that thegenes are operatively linked to transcriptional and translationalsequences. Expression vectors include plasmids, YACs, cosmids,retrovirus, EBV-derived episomes, and all the other vectors that theskilled man will know to be convenient for ensuring the expression ofsaid heavy and/or light chains. The skilled man will realize that thepolynucleotides encoding the heavy and the light chains can be clonedinto different vectors or in the same vector. In a preferred embodiment,said polynucleotides are cloned in the same vector.

Polynucleotides of the invention and vectors comprising these moleculescan be used for the transformation of a suitable mammalian host cell, orany other type of host cell known to the skilled person. Transformationcan be by any known method for introducing polynucleotides into a cellhost. Such methods are well known of the man skilled in the art andinclude dextran-mediated transformation, calcium phosphateprecipitation, polybrene-mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide into liposomes,biolistic injection and direct microinjection of DNA into nuclei.

Antibody Fragments

The antibodies of the present invention include both the full lengthantibodies discussed above, as well as epitope-binding fragments. Asused herein, “antibody fragments” include any portion of an antibodythat retains the ability to bind to the epitope recognized by the fulllength antibody, generally termed “epitope-binding fragments.” Examplesof antibody fragments include, but are not limited to, Fab, Fab′ andF(ab′)₂, Fd, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (dsFv) and fragments comprising either a V_(L) orV_(H) region. Epitope-binding fragments, including single-chainantibodies, may comprise the variable region(s) alone or in combinationwith the entirety or a portion of the following: hinge region, C_(H)1,C_(H)2, and C_(H)3 domains.

Such fragments may contain one or both Fab fragments or the F(ab′)₂fragment. Preferably, the antibody fragments contain all six CDRs of thewhole antibody, although fragments containing fewer than all of suchregions, such as three, four or five CDRs, are also functional. Further,the fragments may be or may combine members of any one of the followingimmunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and the subclassesthereof.

Fab and F(ab′)₂ fragments may be produced by proteolytic cleavage, usingenzymes such as papain (Fab fragments) or pepsin (F(ab′)₂ fragments).

The “single-chain FVs” (“scFvs”) fragments are epitope-binding fragmentsthat contain at least one fragment of an antibody heavy chain variableregion (V_(H)) linked to at least one fragment of an antibody lightchain variable region (V_(L)). The linker may be a short, flexiblepeptide selected to ensure that the proper three-dimensional folding ofthe (V_(L)) and (V_(H)) regions occurs once they are linked so as tomaintain the target molecule binding-specificity of the whole antibodyfrom which the single-chain antibody fragment is derived. The carboxylterminus of the (V_(L)) or (V_(H)) sequence may be covalently linked bya linker to the amino acid terminus of a complementary (V_(L)) or(V_(H)) sequence.

Single-chain antibody fragments of the present invention contain aminoacid sequences having at least one of the variable or complementaritydetermining regions (CDRs) of the whole antibodies described in thisspecification, but are lacking some or all of the constant domains ofthose antibodies. These constant domains are not necessary for antigenbinding, but constitute a major portion of the structure of wholeantibodies. Single-chain antibody fragments may therefore overcome someof the problems associated with the use of antibodies containing a partor all of a constant domain. For example, single-chain antibodyfragments tend to be free of undesired interactions between biologicalmolecules and the heavy-chain constant region, or other unwantedbiological activity. Additionally, single-chain antibody fragments areconsiderably smaller than whole antibodies and may therefore havegreater capillary permeability than whole antibodies, allowingsingle-chain antibody fragments to localize and bind to targetantigen-binding sites more efficiently. Also, antibody fragments can beproduced on a relatively large scale in prokaryotic cells, thusfacilitating their production. Furthermore, the relatively small size ofsingle-chain antibody fragments makes them less likely to provoke animmune response in a recipient than whole antibodies.

Single-chain antibody fragments may be generated by molecular cloning,antibody phage display library or similar techniques well known to theskilled artisan. These proteins may be produced, for example, ineukaryotic cells or prokaryotic cells, including bacteria. Theepitope-binding fragments of the present invention can also be generatedusing various phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.In particular, such phage can be utilized to display epitope-bindingdomains expressed from a repertoire or combinatorial antibody library(e.g., human or murine). Phage expressing an epitope-binding domain thatbinds the antigen of interest can be selected or identified withantigen, e.g., using labeled antigen bound or captured to a solidsurface or bead. Phage used in these methods are typically filamentousphage including fd and M13 binding domains expressed from phage withFab, Fv or disulfide-stabilized Fv antibody domains recombinantly fusedto either the phage gene III or gene VIII protein.

Examples of phage display methods that can be used to make theepitope-binding fragments of the present invention include thosedisclosed in Brinkman et al., 1995, J. Immunol. Methods, 182: 41-50;Ames et al., 1995, J. Immunol. Methods, 184: 177-186; Kettleborough etal., 1994, Eur. J. Immunol., 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology, 57: 191-280; PCTapplication No. PCT/GB91/01134; PCT publications WO 90/02809; WO91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which isincorporated herein by reference in its entirety.

After phage selection, the regions of the phage encoding the fragmentscan be isolated and used to generate the epitope-binding fragmentsthrough expression in a chosen host, including mammalian cells, insectcells, plant cells, yeast, and bacteria, using recombinant DNAtechnology, e.g., as described in detail below. For example, techniquesto recombinantly produce Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., 1992, BioTechniques, 12(6):864-869; Sawai et al., 1995, AJRI, 34: 26-34; and Better et al., 1988,Science, 240: 1041-1043; said references incorporated by reference intheir entireties. Examples of techniques which can be used to producesingle-chain Fvs and antibodies include those described in U.S. Pat.Nos. 4,946,778 and 5,258,498; Huston et al., 1991, Methods in Enzymology203: 46-88; Shu et al., 1993, Proc. Natl. Acad. Sci. U.S.A., 90:7995-7999; Skerra et al., 1988, Science, 240: 1038-1040.

Functional Equivalents

Also included within the scope of the invention are functionalequivalents of the anti-EphA antibody and the humanized anti-EphA2receptor antibody. The term “functional equivalents” includes antibodieswith homologous sequences, chimeric antibodies, artificial antibodiesand modified antibodies, for example, wherein each functional equivalentis defined by its ability to bind to EphA2 receptor. The skilled artisanwill understand that there is an overlap in the group of moleculestermed “antibody fragments” and the group termed “functionalequivalents.” Methods of producing functional equivalents are known tothe person skilled in the art and are disclosed, for example, in PCTApplication WO 93/21319, European Patent No. EP 0239400; PCT ApplicationWO 89/09622; European Patent No. EP 0338745; and European PatentApplication EP 0332424, which are incorporated in their respectiveentireties by reference.

Antibodies with homologous sequences are those antibodies with aminoacid sequences that have sequence homology with amino acid sequence ofan anti-EphA antibody and a humanized anti-EphA antibody of the presentinvention. Preferably homology is with the amino acid sequence of thevariable regions of the anti-EphA antibody and humanized anti-EphAantibody of the present invention. “Sequence homology” as applied to anamino acid sequence herein is defined as a sequence with at least about90%, 91%, 92%, 93%, or 94% sequence homology, and more preferably atleast about 95%, 96%, 97%, 98%, or 99% sequence homology to anotheramino acid sequence, as determined, for example, by the FASTA searchmethod in accordance with Pearson and Lipman, 1988, Proc. Natl. Acad.Sci. U.S.A., 85: 2444-2448.

A chimeric antibody is one in which different portions of an antibodyare derived from different animal species. For example, an antibodyhaving a variable region derived from a murine monoclonal antibodypaired with a human immunoglobulin constant region. Methods forproducing chimeric antibodies are known in the art. See, e.g., Morrison,1985, Science, 229: 1202; Oi et al., 1986, Bio Techniques, 4: 214;Gillies et al., 1989, J. Immunol. Methods, 125: 191-202; U.S. Pat. Nos.5,807,715; 4,816,567; and 4,816,397, which are incorporated herein byreference in their entireties.

Humanized forms of chimeric antibodies are made by substituting thecomplementarity determining regions of, for example, a mouse antibody,into a human framework domain, e.g., see PCT Pub. No. WO92/22653.Humanized chimeric antibodies preferably have constant regions andvariable regions other than the complementarity determining regions(CDRs) derived substantially or exclusively from the corresponding humanantibody regions and CDRs derived substantially or exclusively from amammal other than a human.

Artificial antibodies include scFv fragments, diabodies, triabodies,tetrabodies and mru (see reviews by Winter, G. and Milstein, C., 1991,Nature, 349: 293-299; Hudson, P. J., 1999, Current Opinion inImmunology, 11: 548-557), each of which has antigen-binding ability. Inthe single chain Fv fragment (scFv), the V_(H) and V_(L) domains of anantibody are linked by a flexible peptide. Typically, this linkerpeptide is about 15 amino acid residues long. If the linker is muchsmaller, for example 5 amino acids, diabodies are formed, which arebivalent scFv dimers. If the linker is reduced to less than three aminoacid residues, trimeric and tetrameric structures are formed that arecalled triabodies and tetrabodies. The smallest binding unit of anantibody is a CDR, typically the CDR2 of the heavy chain which hassufficient specific recognition and binding that it can be usedseparately. Such a fragment is called a molecular recognition unit ormru. Several such mrus can be linked together with short linkerpeptides, therefore forming an artificial binding protein with higheravidity than a single mru.

The functional equivalents of the present application also includemodified antibodies, e.g., antibodies modified by the covalentattachment of any type of molecule to the antibody. For example,modified antibodies include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Thecovalent attachment does not prevent the antibody from generating ananti-idiotypic response. These modifications may be carried out by knowntechniques, including, but not limited to, specific chemical cleavage,acetylation, formylation, metabolic synthesis of tunicamycin, etc.Additionally, the modified antibodies may contain one or morenon-classical amino acids.

Functional equivalents may be produced by interchanging different CDRson different chains within different frameworks. Thus, for example,different classes of antibody are possible for a given set of CDRs bysubstitution of different heavy chains, whereby, for example, IgG1-4,IgM, IgA1-2, IgD, IgE antibody types and isotypes may be produced.Similarly, artificial antibodies within the scope of the invention maybe produced by embedding a given set of CDRs within an entirelysynthetic framework.

Functional equivalents may be readily produced by mutation, deletionand/or insertion within the variable and/or constant region sequencesthat flank a particular set of CDRs, using a wide variety of methodsknown in the art.

The antibody fragments and functional equivalents of the presentinvention encompass those molecules with a detectable degree of bindingto EphA, when compared to the 37.3D7, 37.1F5, 53.2H11, EphA2-N1 orEphA2-N2 antibody. A detectable degree of binding includes all values inthe range of at least 10-100%, preferably at least 50%, 60% or 70%, morepreferably at least 75%, 80%, 85%, 90%, 95% or 99% the binding abilityof the murine 37.3D7, 37.1F5, 53.2H11, EphA2-N1 or EphA2-N2 antibody toEphA.

Improved Antibodies

The CDRs are of primary importance for epitope recognition and antibodybinding. However, changes may be made to the residues that comprise theCDRs without interfering with the ability of the antibody to recognizeand bind its cognate epitope. For example, changes that do not affectepitope recognition, yet increase the binding affinity of the antibodyfor the epitope may be made.

Thus, also included in the scope of the present invention are improvedversions of both the murine and humanized antibodies, which alsospecifically recognize and bind EphA, preferably with increasedaffinity.

Several studies have surveyed the effects of introducing one or moreamino acid changes at various positions in the sequence of an antibody,based on the knowledge of the primary antibody sequence, on itsproperties such as binding and level of expression (Yang, W. P. et al.,1995, J. Mol. Biol., 254: 392-403; Rader, C. et al., 1998, Proc. Natl.Acad. Sci. U.S.A., 95: 8910-8915; Vaughan, T. J. et al., 1998, NatureBiotechnology, 16: 535-539).

In these studies, equivalents of the primary antibody have beengenerated by changing the sequences of the heavy and light chain genesin the CDR1, CDR2, CDR3, or framework regions, using methods such asoligonucleotide-mediated site-directed mutagenesis, cassettemutagenesis, error-prone PCR, DNA shuffling, or mutator-strains of E.coli (Vaughan, T. J. et al., 1998, Nature Biotechnology, 16: 535-539;Adey, N. B. et al., 1996, Chapter 16, pp. 277-291, in “Phage Display ofPeptides and Proteins”, Eds. Kay, B. K. et al., Academic Press). Thesemethods of changing the sequence of the primary antibody have resultedin improved affinities of the secondary antibodies (Gram, H. et al.,1992, Proc. Natl. Acad. Sci. U.S.A., 89: 3576-3580; Boder, E. T. et al.,2000, Proc. Natl. Acad. Sci. U.S.A., 97: 10701-10705; Davies, J. andRiechmann, L., 1996, Immunotechnolgy, 2: 169-179; Thompson, J. et al.,1996, J. Mol. Biol., 256: 77-88; Short, M. K. et al., 2002, J. Biol.Chem., 277: 16365-16370; Furukawa, K. et al., 2001, J. Biol. Chem., 276:27622-27628).

By a similar directed strategy of changing one or more amino acidresidues of the antibody, the antibody sequences described in thisinvention can be used to develop anti-EphA antibodies with improvedfunctions, including improved affinity for EphA.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, and (4) conferor modify other physico-chemical or functional properties of suchanalogs. Analogs can include various muteins of a sequence other thanthe naturally-occurring peptide sequence. For example, single ormultiple amino acid substitutions (preferably conservative amino acidsubstitutions) may be made in the naturally-occurring sequence(preferably in the portion of the polypeptide outside the domain (s)forming intermolecular contacts. A conservative amino acid substitutionshould not substantially change the structural characteristics of theparent sequence (e. g., a replacement amino acid should not tend tobreak a helix that occurs in the parent sequence, or disrupt other typesof secondary structure that characterizes the parent sequence). Examplesof art-recognized polypeptide secondary and tertiary structures aredescribed in Proteins, Structures and Molecular Principles (Creighton,Ed., W. H. Freeman and Company, New York (1984)); Introduction toProtein Structure (C. Branden and J. Tooze, eds., Garland Publishing,New York, N. Y. (1991)); and Thornton et al., 1991, Nature, 354: 105,which are each incorporated herein by reference.

Improved antibodies also include those antibodies having improvedcharacteristics that are prepared by the standard techniques of animalimmunization, hybridoma formation and selection for antibodies withspecific characteristics.

The present invention also includes cytotoxic conjugates. Thesecytotoxic conjugates comprise two primary components, a cell-bindingagent and a cytotoxic agent.

As used herein, the term “cell binding agent” refers to an agent thatspecifically recognizes and binds the EphA receptors on the cellsurface. In one embodiment, the cell binding agent specificallyrecognizes the EphA receptor such that it allows the conjugates to actin a targeted fashion with little side-effects resulting fromnon-specific binding.

In another embodiment, the cell binding agent of the present inventionalso specifically recognizes the EphA receptor so that the conjugateswill be in contact with the target cell for a sufficient period of timeto allow the cytotoxic drug portion of the conjugate to act on the cell,and/or to allow the conjugates sufficient time in which to beinternalized by the cell.

In a preferred embodiment, the cytotoxic conjugates comprise ananti-EphA antibody as the cell binding agent, more preferably the murine37.3D7, 37.1F5, 53.2H11, EphA2-N1 or EphA2-N2 anti-EphA monoclonalantibody. In a more preferred embodiment, the cytotoxic conjugatecomprises a humanized 37.3D7, 37.1F5, 53.2H11, EphA2-N1 or EphA2-N2antibody or an epitope-binding fragment thereof. The 37.3D7, 37.1F5,53.2H11, EphA2-N1 or EphA2-N2 antibody is able to specifically recognizean EphA receptor, such as EphA2, and directs the cytotoxic agent to anabnormal cell or a tissue, such as cancer cells, in a targeted fashion.

The second component of the cytotoxic conjugates of the presentinvention is a cytotoxic agent. The term “cytotoxic agent” as usedherein refers to a substance that reduces or blocks the function, orgrowth, of cells and/or causes destruction of cells.

In preferred embodiments, the cytotoxic agent is a taxoid, amaytansinoid such as DM1 or DM4, a small drug, a tomaymycin derivative,a leptomycin derivative, a prodrug, CC-1065 or a CC-1065 analog. Inpreferred embodiments, the cell binding agents of the present inventionare covalently attached, directly or via a cleavable or non-cleavablelinker, to the cytotoxic agent.

The cell binding agents, cytotoxic agents, and linkers are discussed inmore detail below.

Cell Binding Agents

The effectiveness of the compounds of the present invention astherapeutic agents depends on the careful selection of an appropriatecell binding agent. Cell binding agents may be of any kind presentlyknown, or that become known, and includes peptides and non-peptides. Thecell binding agent may be any compound that can bind a cell, either in aspecific or non-specific manner. Generally, these can be antibodies(especially monoclonal antibodies), lymphokines, hormones, growthfactors, vitamins, nutrient-transport molecules (such as transferrin),or any other cell binding molecule or substance.

More specific examples of cell binding agents that can be used include:

-   -   polyclonal antibodies;    -   monoclonal antibodies;    -   fragments of antibodies such as Fab, Fab′, and F(ab′)₂, Fv        (Parham, 1983, J. Immunol., 131:2895-2902; Spring et al.,        1974, J. Immunol., 113: 470-478; Nisonoff et al., 1960, Arch.        Biochem. Biophys., 89: 230-244).

Preferably, a humanized anti-EphA antibody is used as the cell bindingagent of the present invention. More preferably the humanized anti-EphAantibody is selected from humanized or resurfaced 37.3D7, 37.1F5;53.2H11; EphA2-N1 and EphA2-N2 antibodies.

Cytotoxic Agents

In another embodiment, the humanized antibody or an epitope-bindingfragment thereof can be conjugated to a drug, such as a maytansinoid ora tomaymycin derivative, to form a prodrug having specific cytotoxicitytowards antigen-expressing cells by targeting the drug to the EphA2receptor. Cytotoxic conjugates comprising such antibodies and a small,highly toxic drug (e.g., maytansinoids, taxanes, tomaymycin derivatives,a leptomycin derivative, CC-1065, and CC-1065 analogs) can be used as atherapeutic for treatment of tumors, such as breast and ovarian tumors.

The cytotoxic agent used in the cytotoxic conjugate of the presentinvention may be any compound that results in the death of a cell, orinduces cell death, or in some manner decreases cell viability.Preferred cytotoxic agents include, for example, maytansinoids andmaytansinoid analogs, a prodrug, tomaymycin derivatives, taxoids, aleptomycin derivative, CC-1065 and CC-1065 analogs, defined below. Thesecytotoxic agents are conjugated to the antibodies, antibodies fragments,functional equivalents, improved antibodies and their analogs asdisclosed herein.

The cytotoxic conjugates may be prepared by in vitro methods. In orderto link a drug or prodrug to the antibody, a linking group is used.Suitable linking groups are well known in the art and include disulfidegroups, thioether groups, acid labile groups, photolabile groups,peptidase labile groups and esterase labile groups. Preferred linkinggroups are disulfide groups and thioether groups. For example,conjugates can be constructed using a disulfide exchange reaction or byforming a thioether bond between the antibody and the drug or prodrug.

Maytansinoids

Among the cytotoxic agents that may be used in the present invention toform a cytotoxic conjugate, are maytansinoids and maytansinoid analogs.Examples of suitable maytansinoids include maytansinol and maytansinolanalogs. Maytansinoids are drugs that inhibit microtubule formation andthat are highly toxic to mammalian cells.

Examples of suitable maytansinol analogues include those having amodified aromatic ring and those having modifications at otherpositions. Such suitable maytansinoids are disclosed in U.S. Pat. Nos.4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929;4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348;4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.

Specific examples of suitable analogues of maytansinol having a modifiedaromatic ring include:

(1) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by LAH reductionof ansamytocin P2);

(2) C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and

(3) C-20-demethoxy, C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No.4,294,757) (prepared by acylation using acyl chlorides).

Specific examples of suitable analogues of maytansinol havingmodifications of other positions include:

(1) C-9-SH (U.S. Pat. No. 4,424,219) (prepared by the reaction ofmaytansinol with H₂S or P₂S₅),

(2) C-14-alkoxymethyl (demethoxy/CH₂OR) (U.S. Pat. No. 4,331,598);

(3) C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia);

(4) C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by theconversion of maytansinol by Streptomyces);

(5) C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudiflora);

(6) C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (preparedby the demethylation of maytansinol by Streptomyces); and

(7) 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by the titaniumtrichloride/LAH reduction of maytansinol).

In a preferred embodiment, the cytotoxic conjugates of the presentinvention utilize the thiol-containing maytansinoid (DM1), formallytermed N^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine, asthe cytotoxic agent. DM1 is represented by the following structuralformula (I):

In another preferred embodiment, the cytotoxic conjugates of the presentinvention utilize the thiol-containing maytansinoidN^(2′)-deacetyl-N-^(2′)(4-methyl-4-mercapto-1-oxopentyl)-maytansine asthe cytotoxic agent. DM4 is represented by the following structuralformula (II):

In further embodiments of the invention, other maytansines, includingthiol and disulfide-containing maytansinoids bearing a mono or di-alkylsubstitution on the carbon atom bearing the sulfur atom, may be used.These include a maytansinoid having, at C-3, C-14 hydroxymethyl, C-15hydroxy, or C-20 desmethyl, an acylated amino acid side chain with anacyl group bearing a hindered sulfhydryl group, wherein the carbon atomof the acyl group bearing the thiol functionality has one or twosubstituents, said substituents being CH₃, C₂H₅, linear or branchedalkyl or alkenyl having from 1 to 10 carbon atoms, cyclic alkyl oralkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl, orheterocyclic aromatic or heterocycloalkyl radical, and further whereinone of the substituents can be H, and wherein the acyl group has alinear chain length of at least three carbon atoms between the carbonylfunctionality and the sulfur atom.

Such additional maytansines include compounds represented by formula(III):

wherein:

Y′ represents

(CR₇R₈)_(l)(CR₉═CR₁₀)_(p)(C≡C)_(q)A_(r)(CR₅R₆)_(m)D_(u)(CR₁₁═CR₁₂)_(r)(C≡C)_(s)B_(t)(CR₃R₄)_(n)CR₁R₂SZ,

wherein:

-   -   R₁ and R₂ are each independently CH₃, C₂H₅, linear alkyl or        alkenyl having from 1 to 10 carbon atoms, branched or cyclic        alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl,        substituted phenyl or heterocyclic aromatic or heterocycloalkyl        radical, and in addition R₂ can be H;    -   A, B, D are cycloalkyl or cycloalkenyl having 3-10 carbon atoms,        simple or substituted aryl or heterocyclic aromatic or        heterocycloalkyl radical;    -   R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, and R₁₂ are each        independently H, CH₃, C₂H₅, linear alkyl or alkenyl having from        1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having        from 3 to 10 carbon atoms, phenyl, substituted phenyl or        heterocyclic aromatic or heterocycloalkyl radical;    -   l, m, n, o, p, q, r, s, and t are each independently 0 or an        integer of from 1 to 5, provided that at least two of l, m, n,        o, p, q, r, s and t are not zero at any one time; and    -   Z is H, SR or —COR, wherein R is linear alkyl or alkenyl having        from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl        having from 3 to 10 carbon atoms, or simple or substituted aryl        or heterocyclic aromatic or heterocycloalkyl radical.

Preferred embodiments of formula (III) include compounds of formula(III) wherein:

-   -   R₁ is methyl, R₂ is H and Z is H.    -   R₁ and R₂ are methyl and Z is H.    -   R₁ is methyl, R₂ is H, and Z is —SCH₃    -   R₁ and R₂ are methyl, and Z is —SCH₃

Such additional maytansines also include compounds represented byformula (IV-L), (IV-D), or (IV-D,L):

wherein:

-   -   Y represents (CR₇R₈)_(l)(CR₅R₆)_(m)(CR₃R₄)_(n)CR₁R₂SZ,

wherein:

-   -   R₁ and R₂ are each independently CH₃, C₂H₅, linear alkyl or        alkenyl having from 1 to 10 carbon atoms, branched or cyclic        alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl,        substituted phenyl, or heterocyclic aromatic or heterocycloalkyl        radical, and in addition R₂ can be H;    -   R₃, R₄, R₅, R₆, R₇ and R₈ are each independently H, CH₃, C₂H₅,        linear alkyl or alkenyl having from 1 to 10 carbon atoms,        branched or cyclic alkyl or alkenyl having from 3 to 10 carbon        atoms, phenyl, substituted phenyl, or heterocyclic aromatic or        heterocycloalkyl radical;    -   l, m and n are each independently an integer of from 1 to 5, and        in addition n can be 0;    -   Z is H, SR or —COR wherein R is linear or branched alkyl or        alkenyl having from 1 to 10 carbon atoms, cyclic alkyl or        alkenyl having from 3 to 10 carbon atoms, or simple or        substituted aryl or heterocyclic aromatic or heterocycloalkyl        radical; and    -   May represents a maytansinoid which bears the side chain at C-3,        C-14 hydroxymethyl, C-15 hydroxy or C-20 desmethyl.

Preferred embodiments of formulas (IV-L), (IV-D) and (IV-D,L) includecompounds of formulas (IV-L), (IV-D) and (IV-D,L) wherein:

-   -   R₁ is methyl, R₂ is H, R₃, R₆, R₇, and R₈ are each H, l and m        are each 1, n is 0, and Z is H.    -   R₁ and R₂ are methyl, R₅, R₆, R₇, R₈ are each H, l and m are 1,        n is 0, and Z is H.    -   R₁ is methyl, R₂ is H, R₅, R₆, R₇, and R₈ are each H, l and m        are each 1, n is 0, and Z is —SCH₃.    -   R₁ and R₂ are methyl, R₅, R₆, R₇, R₈ are each H, l and m are 1,        n is 0, and Z is —SCH₃.

Preferably the cytotoxic agent is represented by formula (IV-L).

Such additional maytansines also include compounds represented byformula (V):

wherein:

Y represents (CR₇R₈)_(l)(CR₅R₆)_(m)(CR₃R₄)_(n)CR₁R₂SZ,

wherein:

-   -   R₁ and R₂ are each independently CH₃, C₂H₅, linear alkyl or        alkenyl having from 1 to 10 carbon atoms, branched or cyclic        alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl,        substituted phenyl or heterocyclic aromatic or heterocycloalkyl        radical, and in addition R₂ can be H;    -   R₃, R₄, R₅, R₆, R₇ and R₈ are each independently H, CH₃, C₂H₅,        linear alkyl or alkenyl having from 1 to 10 carbon atoms,        branched or cyclic alkyl or alkenyl having from 3 to 10 carbon        atoms, phenyl, substituted phenyl, or heterocyclic aromatic or        heterocycloalkyl radical;    -   l, m and n are each independently an integer of from 1 to 5, and        in addition n can be 0; and    -   Z is H, SR or —COR, wherein R is linear alkyl or alkenyl having        from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl        having from 3 to 10 carbon atoms, or simple or substituted aryl        or heterocyclic aromatic or heterocycloalkyl radical.

Preferred embodiments of formula (V) include compounds of formula (V)wherein:

-   -   R₁ is methyl, R₂ is H, R5, R6, R7, and R8 are each H; l and m        are each 1; n is 0; and Z is H.    -   R₁ and R₂ are methyl; R₅, R₆, R₇, R₈ are each H, l and m are 1;        n is 0; and Z is H.    -   R₁ is methyl, R₂ is H, R₅, R₆, R₇, and R₈ are each H, l and m        are each 1, n is 0, and Z is —SCH₃.    -   R₁ and R₂ are methyl, R₅, R₆, R₇, R₈ are each H, l and m are 1,        n is 0, and Z is —SCH₃.

Such additional maytansines further include compounds represented byformula (VI-L), (VI-D), or (VI-D,L):

wherein:

-   -   Y₂ represents (CR₇R₈)_(l)(CR₅R₆)_(m)(CR₃R₄)_(n)CR₁R₂SZ₂,

wherein:

-   -   R₁ and R₂ are each independently CH₃, C₂H₅, linear alkyl or        alkenyl having from 1 to 10 carbon atoms, branched or cyclic        alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl,        substituted phenyl or heterocyclic aromatic or heterocycloalkyl        radical, and in addition R₂ can be H;    -   R₃, R₄, R₅, R₆, R₇ and R₈ are each independently H, CH₃, C₂H₅,        linear cyclic alkyl or alkenyl having from 1 to 10 carbon atoms,        branched or cyclic alkyl or alkenyl having from 3 to 10 carbon        atoms, phenyl, substituted phenyl or heterocyclic aromatic or        heterocycloalkyl radical;    -   l, m and n are each independently an integer of from 1 to 5, and        in addition n can be 0;    -   Z₂ is SR or COR, wherein R is linear alkyl or alkenyl having        from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl        having from 3 to 10 carbon atoms, or simple or substituted aryl        or heterocyclic aromatic or heterocycloalkyl radical; and    -   May is a maytansinoid.

Such additional maytansines also include compounds represented byformula (VII):

wherein:

Y₂′ represents

(CR₇R₈)_(l)(CR₉═CR₁₀)_(p)(C≡C)_(q)A_(r)(CR₅R₆)_(m)D_(u)(CR₁₁═CR₁₂)_(r)(C≡C)_(s)B_(t)(CR₃R₄)_(n)CR₁R₂SZ₂,

wherein:

-   -   R₁ and R₂ are each independently CH₃, C₂H₅, linear branched or        alkyl or alkenyl having from 1 to 10 carbon atoms, cyclic alkyl        or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted        phenyl or heterocyclic aromatic or heterocycloalkyl radical, and        in addition R₂ can be H;    -   A, B, and D each independently is cycloalkyl or cycloalkenyl        having 3 to 10 carbon atoms, simple or substituted aryl, or        heterocyclic aromatic or heterocycloalkyl radical;    -   R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, and R₁₂ are each        independently H, CH₃, C₂H₅, linear alkyl or alkenyl having from        1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having        from 3 to 10 carbon atoms, phenyl, substituted phenyl or        heterocyclic aromatic or heterocycloalkyl radical;    -   l, m, n, o, p, q, r, s, and t are each independently 0 or an        integer of from 1 to 5, provided that at least two of l, m, n,        o, p, q, r, s and t are not zero at any one time; and    -   Z₂ is SR or —COR, wherein R is linear alkyl or alkenyl having        from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl        having from 3-10 carbon atoms, or simple or substituted aryl or        heterocyclic aromatic or heterocycloalkyl radical.

Preferred embodiments of formula (VII) include compounds of formula(VII) wherein: R, is methyl, R₂ is H.

The above-mentioned maytansinoids can be conjugated to anti-EphAantibody 37.3D7, 37.1F5, 53.2H11, EphA2-N1 or EphA2-N2 or a homologue orfragment thereof, wherein the antibody is linked to the maytansinoidusing the thiol or disulfide functionality that is present on the acylgroup of an acylated amino acid side chain found at C-3, C-14hydroxymethyl, C-15 hydroxy or C-20 desmethyl of the maytansinoid, andwherein the acyl group of the acylated amino acid side chain has itsthiol or disulfide functionality located at a carbon atom that has oneor two substituents, said substituents being CH₃, C₂H₅, linear alkyl oralkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl oralkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl orheterocyclic aromatic or heterocycloalkyl radical, and in addition oneof the substituents can be H, and wherein the acyl group has a linearchain length of at least three carbon atoms between the carbonylfunctionality and the sulfur atom.

A preferred conjugate of the present invention is the one that comprisesthe anti-EphA antibody 37.3D7, 37.1F5, 53.2H11, EphA2-N1 or EphA2-N2 ora homologue or fragment thereof, conjugated to a maytansinoid of formula(VIII):

wherein:

Y₁′ represents

(CR₇R₈)_(l)(CR₉═CR₁₀)_(p)(C≡C)_(q)A_(r)(CR₅R₆)_(m)D_(u)(CR₁₁═CR₁₂)_(r)(C≡C)_(s)B_(t)(CR₃R₄)_(n)CR₁R₂S—,

wherein:

-   -   A, B, and D, each independently is cycloalkyl or cycloalkenyl        having 3-10 carbon atoms, simple or substituted aryl, or        heterocyclic aromatic or heterocycloalkyl radical;    -   R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, and R₁₂ are each        independently H, CH₃, C₂H₅, linear alkyl or alkenyl having from        1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having        from 3 to 10 carbon atoms, phenyl, substituted phenyl or        heterocyclic aromatic or heterocycloalkyl radical; and    -   l, m, n, o, p, q, r, s, and t are each independently 0 or an        integer of from 1 to 5, provided that at least two of l, m, n,        o, p, q, r, s and t are non-not zero at any one time.

Preferably, R₁ is methyl, R₂ is H, or R₁ and R₂ are methyl.

An even more preferred conjugate of the present invention is the onethat comprises the anti-EphA antibody 37.3D7, 37.1F5, 53.2H11, EphA2-N1or EphA2-N2 or a homologue or fragment thereof, conjugated to amaytansinoid of formula (IX-L), (IX-D), or (IX-D,L):

wherein:

Y₁ represents (CR₇R₈)_(l)(CR₅R₆)_(m)(CR₃R₄)_(n)CR₁R₂S—,

wherein:

-   -   R₁ and R₂ are each independently CH₃, C₂H₅, linear alkyl or        alkenyl having from 1 to 10 carbon atoms, branched or cyclic        alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl,        substituted phenyl, heterocyclic aromatic or heterocycloalkyl        radical, and in addition R₂ can be H;    -   R₃, R₄, R₅, R₆, R₇ and R₈ are each independently H, CH₃, C₂H₅,        linear alkyl or alkenyl having from 1 to 10 carbon atoms,        branched or cyclic alkyl or alkenyl having from 3 to 10 carbon        atoms, phenyl, substituted phenyl or heterocyclic aromatic or        heterocycloalkyl radical;    -   l, m and n are each independently an integer of from 1 to 5, and        in addition n can be 0; and    -   May represents a maytansinol which bears the side chain at C-3,        C-14 hydroxymethyl, C-15 hydroxy or C-20 desmethyl.

Preferred embodiments of formulas (IX-L), (IX-D) and (IX-D,L) includecompounds of formulas (IX-L), (IX-D) and (IX-D,L) wherein:

-   -   R₁ is methyl, R₂ is H, or R₁ and R₂ are methyl,    -   R₁ is methyl, R₂ is H, R₅, R₆, R₇ and R₈ are each H; l and m are        each 1; n is 0,    -   R₁ and R₂ are methyl; R₅, R₆, R₇ and R₈ are each H; l and m are        1; n is 0.

Preferably the cytotoxic agent is represented by formula (IX-L).

A further preferred conjugate of the present invention is the one thatcomprises the anti-EphA antibody 37.3D7, 37.1F5, 53.2H11, EphA2-N1 orEphA2-N2 or a homologue or fragment thereof, conjugated to amaytansinoid of formula (X):

wherein the substituents are as defined for formula (IX) above.

Especially preferred are any of the above-described compounds, whereinR₁ is H, R₂ is methyl, R₅, R₆, R₇ and R₈ are each H, l and m are each 1,and n is 0.

Further especially preferred are any of the above-described compounds,wherein R₁ and R₂ are methyl, R₅, R₆, R₇, R₈ are each H, l and m are 1,and n is 0

Further, the L-aminoacyl stereoisomer is preferred.

Each of the maytansinoids taught in pending U.S. patent application Ser.No. 10/849,136, filed May 20, 2004, may also be used in the cytotoxicconjugate of the present invention. The entire disclosure of U.S. patentapplication Ser. No. 10/849,136 is incorporated herein by reference.

Disulfide-Containing Linking Groups

In order to link the maytansinoid to a cell binding agent, such as the37.3D7, 37.1F5, 53.2H11, EphA2-N1 or EphA2-N2 antibody, the maytansinoidcomprises a linking moiety. The linking moiety contains a chemical bondthat allows for the release of fully active maytansinoids at aparticular site. Suitable chemical bonds are well known in the art andinclude disulfide bonds, acid labile bonds, photolabile bonds, peptidaselabile bonds and esterase labile bonds. Preferred are disulfide bonds.

The linking moiety also comprises a reactive chemical group. In apreferred embodiment, the reactive chemical group can be covalentlybound to the maytansinoid via a disulfide bond linking moiety.

Particularly preferred reactive chemical groups are N-succinimidylesters and N-sulfosuccinimidyl esters.

Particularly preferred maytansinoids comprising a linking moiety thatcontains a reactive chemical group are C-3 esters of maytansinol and itsanalogs where the linking moiety contains a disulfide bond and thechemical reactive group comprises a N-succinimidyl orN-sulfosuccinimidyl ester.

Many positions on maytansinoids can serve as the position to chemicallylink the linking moiety. For example, the C-3 position having a hydroxylgroup, the C-14 position modified with hydroxymethyl, the C-15 positionmodified with hydroxy and the C-20 position having a hydroxy group areall expected to be useful. However the C-3 position is preferred and theC-3 position of maytansinol is especially preferred.

While the synthesis of esters of maytansinol having a linking moiety isdescribed in terms of disulfide bond-containing linking moieties, one ofskill in the art will understand that linking moieties with otherchemical bonds (as described above) can also be used with the presentinvention, as can other maytansinoids. Specific examples of otherchemical bonds include acid labile bonds, photolabile bonds, peptidaselabile bonds and esterase labile bonds. The disclosure of U.S. Pat. No.5,208,020, incorporated herein, teaches the production of maytansinoidsbearing such bonds.

The synthesis of maytansinoids and maytansinoid derivatives having adisulfide moiety that bears a reactive group is described in U.S. Pat.Nos. 6,441,163 and 6,333,410, and U.S. application Ser. No. 10/161,651,each of which is herein incorporated by reference.

The reactive group-containing maytansinoids, such as DM1, are reactedwith an antibody, such as the 37.3D7, 37.1F5, 53.2H11, EphA2-N1 orEphA2-N2 antibody, to produce cytotoxic conjugates. These conjugates maybe purified by HPLC or by gel-filtration.

Several excellent schemes for producing such antibody-maytansinoidconjugates are provided in U.S. Pat. No. 6,333,410, and U.S. applicationSer. Nos. 09/867,598, 10/161,651 and 10/024,290, each of which isincorporated herein in its entirety.

In general, a solution of an antibody in aqueous buffer may be incubatedwith a molar excess of maytansinoids having a disulfide moiety thatbears a reactive group. The reaction mixture can be quenched by additionof excess amine (such as ethanolamine, taurine, etc.). Themaytansinoid-antibody conjugate may then be purified by gel-filtration.

The number of maytansinoid molecules bound per antibody molecule can bedetermined by measuring spectrophotometrically the ratio of theabsorbance at 252 nm and 280 nm. An average of 1-10 maytansinoidmolecules/antibody molecule is preferred.

-   -   Conjugates of antibodies with maytansinoid drugs can be        evaluated for their ability to suppress proliferation of various        unwanted cell lines in vitro. For example, cell lines such as        the human epidermoid carcinoma line A-431, the human small cell        lung cancer cell line SW2, the human breast tumor line SKBR3 and        the Burkitt's lymphoma cell line Namalwa can easily be used for        the assessment of cytotoxicity of these compounds. Cells to be        evaluated can be exposed to the compounds for 24 hours and the        surviving fractions of cells measured in direct assays by known        methods. IC₅₀ values can then be calculated from the results of        the assays.

PEG-Containing Linking Groups

Maytansinoids may also be linked to cell binding agents using PEGlinking groups, as set forth in U.S. application Ser. No. 10/024,290.These PEG linking groups are soluble both in water and in non-aqueoussolvents, and can be used to join one or more cytotoxic agents to a cellbinding agent. Exemplary PEG linking groups include hetero-bifunctionalPEG linkers that bind to cytotoxic agents and cell binding agents atopposite ends of the linkers through a functional sulfhydryl ordisulfide group at one end, and an active ester at the other end.

As a general example of the synthesis of a cytotoxic conjugate using aPEG linking group, reference is again made to U.S. application Ser. No.10/024,290 for specific details. Synthesis begins with the reaction ofone or more cytotoxic agents bearing a reactive PEG moiety with acell-binding agent, resulting in displacement of the terminal activeester of each reactive PEG moiety by an amino acid residue of the cellbinding agent, such as the 37.3D7, 37.1F5, 53.2H11, EphA2-N1 or EphA2-N2antibody, to yield a cytotoxic conjugate comprising one or morecytotoxic agents covalently bonded to a cell binding agent through a PEGlinking group.

Taxanes

The cytotoxic agent used in the cytotoxic conjugates according to thepresent invention may also be a taxane or derivative thereof.

Taxanes are a family of compounds that includes paclitaxel (taxol), acytotoxic natural product, and docetaxel (Taxotere), a semi-syntheticderivative, two compounds that are widely used in the treatment ofcancer. Taxanes are mitotic-spindle poisons that inhibit thedepolymerization of tubulin, resulting in cell death. While docetaxeland paclitaxel are useful agents in the treatment of cancer, theirantitumor activity is limited because of their non-specific toxicitytowards normal cells. Further, compounds like paclitaxel and docetaxelthemselves are not sufficiently potent to be used in conjugates of cellbinding agents.

A preferred taxane for use in the preparation of cytotoxic conjugates isthe taxane of formula (XI):

Methods for synthesizing taxanes that may be used in the cytotoxicconjugates of the present invention, along with methods for conjugatingthe taxanes to a cell binding agent, such as the 37.3D7, 37.1F5,53.2H11, EphA2-N1 or EphA2-N2 antibody, are described in detail in U.S.Pat. Nos. 5,416,064, 5,475,092, 6,340,701, 6,372,738 and 6,436,931, andin U.S. application Ser. Nos. 10/024,290, 10/144,042, 10/207,814,10/210,112 and 10/369,563.

Tomaymycin Derivatives

The cytotoxic according to the present invention may also a tomaymycinderivative. Tomaymycin derivatives are pyrrolo[1,4]benzodiazepines(PBDs), a known class of compounds exerting their biological propertiesby covalently binding to the N2 of guanine in the minor groove of DNA.PBDs include a number of minor groove binders such as anthramycin,neothramycin and DC-81.

Novel tomaymycin derivatives that retain high cytotoxicity and that canbe effectively linked to cell binding agents are described in theInternational Application No. PCT/IB2007/000142, whose content is hereinincorporated by reference. The cell binding agent-tomaymycin derivativecomplexes permit the full measure of the cytotoxic action of thetomaymycin derivatives to be applied in a targeted fashion againstunwanted cells only, therefore avoiding side effects due to damage tonon-targeted healthy cells.

The cytotoxic agent according to the present invention comprises one ormore tomaymycin derivatives, linked to a cell binding agent, such as the37.3D7, 37.1F5, 53.2H11, EphA2-N1 or EphA2-N2 antibody, via a linkinggroup. The linking group is part of a chemical moiety that is covalentlybound to a tomaymycin derivative through conventional methods. In apreferred embodiment, the chemical moiety can be covalently bound to thetomaymycin derivative via a disulfide bond.

The tomaymycin derivatives useful in the present invention have theformula (XII) shown below:

wherein

---- represents an optional single bond;

represents either a single bond or a double bond;

provided that when

represents a single bond, U and U′, the same or different, independentlyrepresent H, and W and W′, the same or different, are independentlyselected from the group consisting of OH, an ether such as —OR, an ester(e.g. an acetate), such as —OCOR, a carbonate such as —OCOOR, acarbamate such as —OCONRR′, a cyclic carbamate, such that N10 and C11are a part of the cycle, a urea such as —NRCONRR′, a thiocarbamate suchas —OCSNHR, a cyclic thiocarbamate such that N10 and C11 are a part ofthe cycle, —SH, a sulfide such as —SR, a sulphoxide such as —SOR, asulfone such as —SOOR, a sulphonate such as —SO3-, a sulfonamide such as—NRSOOR, an amine such as —NRR′, optionally cyclic amine such that N10and C11 are a part of the cycle, a hydroxylamine derivative such as—NROR′, an amide such as —NRCOR, an azido such as —N3, a cyano, a halo,a trialkyl or triarylphosphonium, an aminoacid-derived group; PreferablyW and W′ are the same or different and are OH, Ome, Oet, NHCONH₂, SMe;

and when

represents a double bond, U and U′ are absent and W and W′ represent H;

-   -   R1, R2, R1′, R2′ are the same or different and independently        chosen from Halide or Alkyl optionally substituted by one or        more Hal, CN, NRR′, CF₃, OR, Aryl, Het, S(O)_(q)R, or R1 and R2        and R1′ and R2′ form together a double bond containing group ═B        and ═B′ respectively.

Preferably, R1 and R2 and R1′ and R2′ form together a double bondcontaining group ═B and ═B′ respectively.

-   -   B and B′ are the same or different and independently chosen from        Alkenyl being optionally substituted by one or more Hal, CN,        NRR′, CF₃, OR, Aryl, Het, S(O)_(q)R or B and B′ represent an        oxygen atom.    -   Preferably, B═B′.    -   More preferably, B═B′══CH₂ or ═CH—CH₃,    -   X, X′ are the same or different and independently chosen from        one or more —O—, —NR—, —(C═O)—, —S(O)_(q)—.    -   Preferably, X═X′.    -   More preferably, X═X′═O.    -   A, A′ are the same or different and independently chosen from        Alkyl or Alkenyl optionally containing an oxygen, a nitrogen or        a sulfur atom, each being optionally substituted by one or more        Hal, CN, NRR′, CF₃, OR, S(O)_(q)R, Aryl, Het, Alkyl, Alkenyl.    -   Preferably, A=A′.    -   More preferably, A=A′=linear unsubstituted alkyl.    -   Y, Y′ are the same or different and independently chosen from H,        OR;    -   Preferably, Y═Y′.    -   More preferably, Y═Y′=OAlkyl, more preferably OMethyl.    -   T is —NR—, —O—, —S(O)_(q-), or a 4 to 10-membered aryl,        cycloalkyl, heterocyclic or heteroaryl, each being optionally        substituted by one or more Hal, CN, NRR′, CF₃, R, OR, S(O)_(q)R,        and/or linker(s), or a branched Alkyl, optionally substituted by        one or more Hal, CN, NRR′, CF₃, OR, S(O)_(q)R and/or linker(s),        or a linear Alkyl substituted by one or more Hal, CN, NRR′, CF₃,        OR, S(O)_(q)R and/or linker(s).

Preferably, T is a 4 to 10-membered aryl or heteroaryl, more preferablyphenyl or pyridyl, optionally substituted by one or more linker(s).

Said linker comprises a linking group. Suitable linking groups are wellknown in the art and include thiol, sulfide, disulfide groups, thioethergroups, acid labile groups, photolabile groups, peptidase labile groupsand esterase labile groups. Preferred are disulfide groups and thioethergroups.

When the linking group is a thiol-, sulfide (or so-called thioether —S—)or disulfide (—S—S—)— containing group, the side chain carrying thethiol, the sulfide or disulfide group can be linear or branched,aromatic or heterocyclic. One of ordinary skill in the art can readilyidentify suitable side chains.

Preferably, said linker is of formula:

-G-D-(Z)p-S—Z′

where

G is a single or double bond, —O—, —S— or —NR—;

D is a single bond or -E-, -E-NR—, -E-NR—F—, -E-O—, -E-O—F—, -E-NR—CO—,-E-NR—CO—F—, -E-CO—, —CO-E-, -E-CO—F, -E-S—, -E-S—F—, -E-NR—C—S—,-E-NR—CS—F—;

where E and F are the same or different and are independently chosenfrom linear or branched —(OCH2CH2)iAlkyl(OCH2CH2)j-,-Alkyl(OCH2CH2)i-Alkyl-, —(OCH2CH2)i-, —(OCH2CH2)iCycloalkyl(OCH2CH2)j-,—(OCH2CH2)iHeterocyclic(OCH2CH2)j-, —(OCH2CH2)iAryl(OCH2CH2)j-,—(OCH2CH2)iHeteroaryl(OCH2CH2)j-, -Alkyl-(OCH2CH2)iAlkyl(OCH2CH2)j-,-Alkyl-(OCH2CH2)i-, -Alkyl-(OCH2CH2)iCycloalkyl(OCH2CH2)j-,-Alkyl(OCH2CH2)iHeterocyclic(OCH2CH2)j-,-Alkyl-(OCH2CH2)iAryl(OCH2CH2)j-, -Alkyl(OCH2CH2)iHeteroaryl(OCH2CH2)j-,-Cycloalkyl-Alkyl-, -Alkyl-Cycloalkyl-, -Heterocyclic-Alkyl-,-Alkyl-Heterocyclic-, -Alkyl-Aryl-, -Aryl-Alkyl-, -Alkyl-Heteroaryl-,-Heteroaryl-Alkyl-;

where i and j, identical or different are integers and independentlychosen from 0, 1 to 2000;

Z is linear or branched -Alkyl-;

p is 0 or 1;

Z′ represents H, a thiol protecting group such as COR, R20 or SR20,wherein R20 represents H, methyl, Alkyl, optionally substitutedCycloalkyl, aryl, heteroaryl or heterocyclic, provided that when Z′ isH, said compound is in equilibrium with the corresponding compoundformed by intramolecular cyclisation resulting from addition of thethiol group —SH on the imine bond —NH═ of one of the PBD moieties.

-   -   n, n′, equal or different are 0 or 1.    -   q is 0, 1 or 2.    -   R, R′ are equal or different and independently chosen from H,        Alkyl, Aryl, each being optionally substituted by Hal, CN, NRR′,        CF3, R, OR, S(O)qR, Aryl, Het;

or their pharmaceutically acceptable salts, hydrates, or hydrated salts,or the polymorphic crystalline structures of these compounds or theiroptical isomers, racemates, diastereomers or enantiomers.

The compounds of the general formula (XII) having geometrical andstereoisomers are also a part of the invention.

The N-10, C-11 double bond of tomaymycin derivatives of formula (XII) isknown to be readily convertible in a reversible manner to correspondingimine adducts in the presence of water, an alcohol, a thiol, a primaryor secondary amine, urea and other nucleophiles. This process isreversible and can easily regenerate the corresponding tomaymycinderivatives in the presence of a dehydrating agent, in a non-proticorganic solvant, in vacuum or at high temperatures (Z. Tozuka, 1983, J.Antibiotics, 36: 276).

Thus, reversible derivatives of tomaymycin derivatives of generalformula (XIII) can also be used in the present invention:

where A, X, Y, n, T, A′, X′, Y′, n′, R1, R2, R1′, R2′ are defined as informula (XII) and W, W′ are the same or different and are selected fromthe group consisting of OH, an ether such as —OR, an ester (e.g. anacetate), such as —OCOR, —COOR, a carbonate such as —OCOOR, a carbamatesuch as —OCONRR′, a cyclic carbamate, such that N10 and C11 are a partof the cycle, a urea such as —NRCONRR′, a thiocarbamate such as —OCSNHR,a cyclic thiocarbamate such that N10 and C11 are a part of the cycle,—SH, a sulfide such as —SR, a sulphoxide such as —SOR, a sulfone such as—SOOR, a sulphonate such as —SO3-, a sulfonamide such as —NRSOOR, anamine such as —NRR′, optionally cyclic amine such that N10 and C11 are apart of the cycle, a hydroxylamine derivative such as —NROR′, an amidesuch as —NRCOR, —NRCONRR′, an azido such as —N3, a cyano, a halo, atrialkyl or triarylphosphonium, an aminoacid-derived group. Preferably,W and W′ are the same or different and are OH, Ome, Oet, NHCONH2, SMe.

Compounds of formula (XIII) may thus be considered as solvates,including water when the solvent is water; these solvates can beparticularly useful.

In a preferred embodiment, the tomaymycin derivatives of the inventionare selected from the group consisting in:

-   8,8′-[1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[5-methoxy-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[1,5-pentanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[1,4-butanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[3-methyl-1,5-pentanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[2,6-pyridinediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[4-(3-tert-butoxycarbonylaminopropyloxy)-2,6-pyridinediylbis-(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[5-(3-aminopropyloxy)-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[5-(N-methyl-3-tert-butoxycarbonylaminopropyl)-1,3-benzenediylbis-(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-{5-[3-(4-methyl-4-methyldisulfanyl-pentanoylamino)propyloxy]-1,3-benzenediylbis(methyleneoxy)}-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[5-acetylthiomethyl-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-methylene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   bis-{2-[(S)-2-methylene-7-methoxy-5-oxo-1,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy]-ethyl}-carbamic    acid tert-butyl ester-   8,8′-[3-(2-acetylthioethyl)-1,5-pentanediylbis(oxy)]-bis[(S)-2-methylene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[5-(N-4-mercapto-4,4-dimethylbutanoyl)amino-1,3-benzenediylbis(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[5-(N-4-methyldithio-4,4-dimethylbutanoyl)-amino-1,3-benzenediylbis(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[5-(N-methyl-N-(2-mercapto-2,2-dimethylethyl)amino-1,3-benzenediyl(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[5-(N-methyl-N-(2-methyldithio-2,2-dimethylethyl)amino-1,3-benzenediyl(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(4-(2-(4-mercapto-4-methyl)-pentanamido-ethoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(1-(2-(4-methyl-4-methyldisulfanyl)-pentanamido-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(4-(3-(4-methyl-4-methyldisulfanyl)-pentanamido-propoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(4-(4-(4-methyl-4-methyldisulfanyl)-pentanamido-butoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(4-(3-[4-(4-methyl-4-methyldisulfanyl-pentanoyl)-piperazin-1-yl]-propyl)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(1-(3-[4-(4-methyl-4-methyldisulfanyl-pentanoyl)-piperazin-1-yl]-propyl)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(4-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-ethoxy}-ethoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(1-(2-{2-[2-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]ethoxy}-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(1-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]ethoxy}-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(4-(2-{2-[2-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]ethoxy}-ethoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(1-(2-[methyl-(2-methyl-2-methyldisulfanyl-propyl)-amino]-ethoxy)-benzene-3,5-di    methyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(4-(3-[methyl-(4-methyl-4-methyldisulfanyl-pentanoyl)-amino]-propyl)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(4-(3-[methyl-(2-methyl-2-methyldisulfanyl-propyl)-amino]propyl)-pyridin-2,6-di    methyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(1-(4-methyl-4-methyldisulfanyl)-pentanamido)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]

as well as the corresponding mercapto derivatives, or theirpharmaceutically acceptable salts, hydrates, or hydrated salts, or thepolymorphic crystalline structures of these compounds or their opticalisomers, racemates, diastereomers or enantiomers.

Preferred compounds are those of formula:

where X, X′, A, A′, Y, Y′, T, n, n′ are defined as above.

The compounds of formula (XII) may be prepared in a number of ways wellknown to those skilled in the art. The compounds can be synthesized, forexample, by application or adaptation of the methods described below, orvariations thereon as appreciated by the skilled artisan. Theappropriate modifications and substitutions will be readily apparent andwell known or readily obtainable from the scientific literature to thoseskilled in the art. In particular, such methods can be found in R. C.Larock, Comprehensive Organic Transformations, Wiley-VCH Publishers,1999.

Methods for synthesizing the tomaymycin derivatives which may be used inthe invention are described in the International Application No.PCT/IB2007/000142. Compounds of the present invention may be prepared bya variety of synthetic routes. The reagents and starting materials arecommercially available, or readily synthesized by well-known techniquesby one of ordinary skill in the arts (see, for example, WO 00/12508, WO00/12507, WO 2005/040170, WO 2005/085260, FR1516743, M. Mori et al.,1986, Tetrahedron, 42: 3793-3806).

The conjugate molecules of the invention may be formed using anytechniques. The tomaymycin derivatives of the invention may be linked toan antibody or other cell binding agent via an acid labile linker, or bya photolabile linker. The derivatives can be condensed with a peptidehaving a suitable sequence and subsequently linked to a cell bindingagent to produce a peptidase labile linker. The conjugates can beprepared to contain a primary hydroxyl group, which can be succinylatedand linked to a cell binding agent to produce a conjugate that can becleaved by intracellular esterases to liberate free derivative.Preferably, the derivatives are synthesized to contain a free orprotected thiol group, and then one or more disulfide orthiol-containing derivatives are each covalently linked to the cellbinding agent via a disulfide bond or a thioether link.

Numerous methods of conjugation are taught in U.S. Pat. No. 5,416,064and U.S. Pat. No. 5,475,092. The tomaymycin derivatives can be modifiedto yield a free amino group and then linked to an antibody or other cellbinding agent via an acid labile linker or a photolabile linker. Thetomaymycin derivatives with a free amino or carboxyl group can becondensed with a peptide and subsequently linked to a cell binding agentto produce a peptidase labile linker. The tomaymycin derivatives with afree hydroxyl group on the linker can be succinylated and linked to acell binding agent to produce a conjugate that can be cleaved byintracellular esterases to liberate free drug. Most preferably, thetomaymycin derivatives are treated to create a free or protected thiolgroup, and then the disulfide- or thiol containing tomaymycin dimers arelinked to the cell binding agent via disulfide bonds.

Preferably, monoclonal antibody- or cell binding agent-tomaymycinderivative conjugates are those that are joined via a disulfide bond, asdiscussed above, that are capable of delivering tomaymycin derivatives.Such cell binding conjugates are prepared by known methods such as bymodifying monoclonal antibodies with succinimidylpyridyl-dithiopropionate (SPDP) (Carlsson et al., 1978, Biochem. J.,173: 723-737). The resulting thiopyridyl group is then displaced bytreatment with thiol-containing tomaymycin derivatives to producedisulfide linked conjugates. Alternatively, in the case of thearyldithio-tomaymycin derivatives, the formation of the cell bindingconjugate is effected by direct displacement of the aryl-thiol of thetomaymycin derivative by sulfhydryl groups previously introduced intoantibody molecules. Conjugates containing 1 to 10 tomaymycin derivativedrugs linked via a disulfide bridge are readily prepared by eithermethod.

More specifically, a solution of the dithio-nitropyridyl modifiedantibody at a concentration of 2.5 mg/ml in 0.05 M potassium phosphatebuffer, at pH 7.5 containing 2 mM EDTA is treated with thethiol-containing tomaymycin derivative (1.3 molar eq./dithiopyridylgroup). The release of thio-nitropyridine from the modified antibody ismonitored spectrophotometrically at 325 nm and is complete in about 16hours. The antibody-tomaymycin derivative conjugate is purified andfreed of unreacted drug and other low molecular weight material by gelfiltration through a column of Sephadex G-25 or Sephacryl S300. Thenumber of tomaymycin derivative moieties bound per antibody molecule canbe determined by measuring the ratio of the absorbance at 230 nm and 275nm. An average of 1-10 tomaymycin derivative molecules/antibody moleculecan be linked via disulfide bonds by this method.

The effect of conjugation on binding affinity towards theantigen-expressing cells can be determined using the methods previouslydescribed by Liu et al., 1996, Proc. Natl. Acad. Sci. U.S.A., 93:8618-8623. Cytotoxicity of the tomaymycin derivatives and their antibodyconjugates to cell lines can be measured by back-extrapolation of cellproliferation curves as described in Goldmacher et al., 1985, J.Immunol., 135: 3648-3651. Cytotoxicity of these compounds to adherentcell lines can be determined by clonogenic assays as described inGoldmacher et al., 1986, J. Cell Biol., 102: 1312-1319.

Leptomycin Derivatives

The cytotoxic according to the present invention may also a leptomycinderivative. According to the present invention, “leptomycin derivatives”refer to members of the leptomycin family as defined in Kalesse et al.(2002, Synthesis 8: 981-1003), and includes: leptomycins, such asleptomycin A and leptomycin B, callystatins, ratjadones such asratjadone A and ratjadone B, anguinomycins such as anguinomycin A, B, C,D, kasusamycins, leptolstatin, leptofuranins, such as leptofuranin A, B,C, D. Derivatives of leptomycin A and B are preferred.

More specifically, the derivatives of the invention are of formula (I):

wherein

Ra and Ra′ are H or -Alk; preferably Ra is -Alk, preferably methyl andRa′ is H;

R17 is alkyl optionally substituted by OR, CN, NRR′, perfluoroalkyl;preferably, R17 is alkyl, more preferably methyl or ethyl;

R9 is alkyl optionally substituted by OR, CN, NRR′, perfluoroalkyl;preferably, R9 is alkyl, more preferably methyl;

X is —O— or —NR—; preferably, X is —NR—;

Y is —U—, —NR—U—, —O—U—, —NR—CO—U—, —U—NR—CO—, —U—CO—, —CO—U—;

preferably, when X is —O—, Y is —U—, —NR—U—, —U—NR—CO—;

where U is chosen from linear or branched -Alk-, -Alk(OCH₂CH₂)_(m)—,—(OCH₂CH₂)_(m)-Alk-, -Alk(OCH₂CH₂)_(m)-Alk-, —(OCH₂CH₂)_(m)—,-Cycloalkyl-, -Heterocyclic-, -Cycloalkyl-Alk-, -Alk-Cycloalkyl-,-Heterocyclic-Alk-, -Alk-Heterocyclic-;

where m is an integer chosen from 1 to 2000;

preferably, U is linear or branched -Alk-,

Z is -Alk-;

n is 0 or 1; preferably n is 0;

T represents H, a thiol protecting group such as Ac, R₁ or SR₁, whereinR₁ represents H, methyl, Alk, Cycloalkyl, optionally substituted aryl orheterocyclic, or T represents

where:

Ra, Ra′, R17, R9, X, Y, Z, n are defined as above;

preferably, T is H or SR₁, wherein R₁ represents Alk, more preferablymethyl;

R, R′ identical or different are H or alkyl;

Alk represents a linear or branched alkyl; preferably Alk represents(—(CH_(2-q)(CH₃)_(q))_(p)— where p represents an integer from 1 to 10;and q represents an integer from 0 to 2; preferably, Alk represents—(CH₂)— ou —C(CH₃)₂—.

or their pharmaceutically acceptable salts, hydrates, or hydrated salts,or the polymorphic crystalline structures of these compounds or theiroptical isomers, racemates, diastereomers or enantiomers.

Preferred compounds may be chosen from:

-   (2-Methylsulfanyl-ethyl)-amid of    (2E,10E,12E,16Z,18E)-(R)-6-Hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic    acid-   Bis-[(2-mercaptoethyl)-amid of    (2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic    acid]-   (2-Mercapto-ethyl)-amid of    (2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic    acid-   (2-Methyldisulfanyl-ethyl)-amid of    (2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic    acid-   (2-Methyl-2-methyldisulfanyl-propyl)-amid of    (2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic    acid-   (2-Mercapto-2-methyl-propyl)-amid of    (2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic    acid

or their pharmaceutically acceptable salts, hydrates, or hydrated salts,or the polymorphic crystalline structures of these compounds or theiroptical isomers, racemates, diastereomers or enantiomers.

In order to link the derivative to a cell-binding agent, the derivativemust include a moiety (linking group) that allows the derivatives to belinked to a cell binding agent via a linkage such as a disulfide bond, asulfide (or called herein thioether) bond, an acid-labile group, aphoto-labile group, a peptidase-labile group, or an esterase-labilegroup. The derivatives are prepared so that they contain a moietynecessary to link the leptomycin derivative to a cell binding agent via,for example, a disulfide bond, a thioether bond, an acid-labile group, aphoto-labile group, a peptidase-labile group, or an esterase-labilegroup. In order to further enhance solubility in aqueous solutions, thelinking group can contain a polyethylene glycol spacer. Preferably, asulfide or disulfide linkage is used because the reducing environment ofthe targeted cell results in cleavage of the sulfide or disulfide andrelease of the derivatives with an associated increase in cytotoxicity.

Compounds of the present invention may be prepared by a variety ofsynthetic routes. The reagents and starting materials are commerciallyavailable, or readily synthesized by well-known techniques by one ofordinary skill in the art. Methods for synthesizing leptomycinderivatives that may be used in the cytotoxic conjugates of the presentinvention, along with methods for conjugating said leptomycinderivatives to cell binding agents such as antibodies, are described indetail in in European Patent Application No. 06290948.6, whose contentis incorporated herein by reference.

CC-1065 Analogues

The cytotoxic agent used in the cytotoxic conjugates according to thepresent invention may also be CC-1065 or a derivative thereof.

CC-1065 is a potent anti-tumor antibiotic isolated from the culturebroth of Streptomyces zelensis. CC-1065 is about 1000-fold more potentin vitro than are commonly used anti-cancer drugs, such as doxorubicin,methotrexate and vincristine (B. K. Bhuyan et al., 1982, Cancer Res.,42, 3532-3537). CC-1065 and its analogs are disclosed in U.S. Pat. Nos.6,372,738, 6,340,701, 5,846,545 and 5,585,499.

The cytotoxic potency of CC-1065 has been correlated with its alkylatingactivity and its DNA-binding or DNA-intercalating activity. These twoactivities reside in separate parts of the molecule. Thus, thealkylating activity is contained in the cyclopropapyrroloindole (CPI)subunit and the DNA-binding activity resides in the two pyrroloindolesubunits.

Although CC-1065 has certain attractive features as a cytotoxic agent,it has limitations in therapeutic use. Administration of CC-1065 to micecaused a delayed hepatotoxicity leading to mortality on day 50 after asingle intravenous dose of 12.5 μg/kg (V. L. Reynolds et al., 1986, J.Antibiotics, XXIX: 319-334). This has spurred efforts to develop analogsthat do not cause delayed toxicity, and the synthesis of simpler analogsmodeled on CC-1065 has been described (M. A. Warpehoski et al., 1988, J.Med. Chem., 31: 590-603).

In another series of analogs, the CPI moiety was replaced by acyclopropabenzindole (CBI) moiety (D. L. Boger et al., 1990, J. Org.Chem., 55: 5823-5833; D. L. Boger et al., 1991, BioOrg. Med. Chem.Lett., 1: 115-120). These compounds maintain the high in vitro potencyof the parental drug, without causing delayed toxicity in mice. LikeCC-1065, these compounds are alkylating agents that bind to the minorgroove of DNA in a covalent manner to cause cell death. However,clinical evaluation of the most promising analogs, Adozelesin andCarzelesin, has led to disappointing results (B. F. Foster et al., 1996,Investigational New Drugs, 13: 321-326; I. Wolff et al., 1996, Clin.Cancer Res., 2: 1717-1723). These drugs display poor therapeutic effectsbecause of their high systemic toxicity.

The therapeutic efficacy of CC-1065 analogs can be greatly improved bychanging the in vivo distribution through targeted delivery to the tumorsite, resulting in lower toxicity to non-targeted tissues, and thus,lower systemic toxicity. In order to achieve this goal, conjugates ofanalogs and derivatives of CC-1065 with cell-binding agents thatspecifically target tumor cells have been described (U.S. Pat. Nos.5,475,092; 5,585,499; 5,846,545). These conjugates typically displayhigh target-specific cytotoxicity in vitro, and exceptional anti-tumoractivity in human tumor xenograft models in mice (R. V. J. Chari et al.,1995, Cancer Res., 55: 4079-4084).

Recently, prodrugs of CC-1065 analogs with enhanced solubility inaqueous medium have been described (European Patent Application No.06290379.4). In these prodrugs, the phenolic group of the alkylatingportion of the molecule is protected with a functionality that rendersthe drug stable upon storage in acidic aqueous solution, and confersincreased water solubility to the drug compared to an unprotectedanalog. The protecting group is readily cleaved in vivo at physiologicalpH to give the corresponding active drug. In the prodrugs described inEP 06290379.4, the phenolic substituent is protected as a sulfonic acidcontaining phenyl carbamate which possesses a charge at physiologicalpH, and thus has enhanced water solubility. In order to further enhancewater solubility, an optional polyethylene glycol spacer can beintroduced into the linker between the indolyl subunit and the cleavablelinkage such as a disulfide group. The introduction of this spacer doesnot alter the potency of the drug.

Methods for synthesizing CC-1065 analogs that may be used in thecytotoxic conjugates of the present invention, along with methods forconjugating the analogs to cell binding agents such as antibodies, aredescribed in detail in EP 06290379.4 (whose content is incorporatedherein by reference) and U.S. Pat. Nos. 5,475,092, 5,846,545, 5,585,499,6,534,660 and 6,586,618 and in U.S. application Ser. Nos. 10/116,053 and10/265,452.

Other Drugs

Drugs such as methotrexate, daunorubicin, doxorubicin, vincristine,vinblastine, melphalan, mitomycin C, chlorambucil, calicheamicin,tubulysin and tubulysin analogs, duocarmycin and duocarmycin analogs,dolastatin and dolastatin analogs are also suitable for the preparationof conjugates of the present invention. The drug molecules can also belinked to the antibody molecules through an intermediary carriermolecule such as serum albumin. Doxarubicin and Danorubicin compounds,as described, for example, in U.S. Pat. No. 6,630,579, may also beuseful cytotoxic agents.

Therapeutic Composition

The invention also relates to a therapeutic composition for thetreatment of a hyperproliferative disorder in a mammal which comprises atherapeutically effective amount of a compound of the invention and apharmaceutically acceptable carrier. In one embodiment saidpharmaceutical composition is for the treatment of cancer, including(but not limited to) the following: carcinoma, including that of thebladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach,cervix, thyroid and skin; including squamous cell carcinoma;hematopoietic tumors of lymphoid lineage, including leukemia, acutelymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma,T-cell lymphoma, Burkitt's lymphoma; hematopoietic tumors of myeloidlineage, including acute and chronic myelogenous leukemias andpromyelocytic leukemia; tumors of mesenchymal origin, includingfibrosarcoma and rhabdomyoscarcoma; other tumors, including melanoma,seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of thecentral and peripheral nervous system, including astrocytoma,neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin,including fibrosarcoma, rhabdomyoscarama, and osteosarcoma; and othertumors, including melanoma, xeroderma pigmentosum, keratoactanthoma,seminoma, thyroid follicular cancer and teratocarcinoma, and othercancers yet to be determined in which EphA is expressed predominantly.In a preferred embodiment, the pharmaceutical compositions of theinvention are used for treatment of cancer of the lung, breast, colon,prostate, kidney, pancreas, ovary, cervix and lymphatic organs,osteosarcoma, synovial carcinoma, a sarcoma, head and neck, a glioma,gastric, liver or other carcinomas in which EphA is expressed. Inparticular, the cancer is a metastatic cancer. In another embodiment,said pharmaceutical composition relates to other disorders such as, forexample, autoimmune diseases, such as systemic lupus, rheumatoidarthritis, and multiple sclerosis; graft rejections, such as renaltransplant rejection, liver transplant rejection, lung transplantrejection, cardiac transplant rejection, and bone marrow transplantrejection; graft versus host disease; viral infections, such as mVinfection, HIV infection, AIDS, etc.; and parasite infections, such asgiardiasis, amoebiasis, schistosomiasis, and others as determined by oneof ordinary skill in the art.

The instant invention provides pharmaceutical compositions comprising:

-   -   an effective amount of an antibody, antibody fragment or        antibody conjugate of the present invention, and    -   a pharmaceutically acceptable carrier, which may be inert or        physiologically active.

As used herein, “pharmaceutically-acceptable carriers” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, and the like that are physiologically compatible. Examples ofsuitable carriers, diluents and/or excipients include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanol,and the like, as well as combination thereof. In many cases, it will bepreferable to include isotonic agents, such as sugars, polyalcohols, orsodium chloride in the composition. In particular, relevant examples ofsuitable carrier include: (1) Dulbecco's phosphate buffered saline, pH˜7.4, containing or not containing about 1 mg/ml to 25 mg/ml human serumalbumin, (2) 0.9% saline (0.9% w/v sodium chloride (NaCl)), and (3) 5%(w/v) dextrose; and may also contain an antioxidant such as tryptamineand a stabilizing agent such as Tween 20.

The compositions herein may also contain a further therapeutic agent, asnecessary for the particular disorder being treated. Preferably, theantibody, antibody fragment or antibody conjugate of the presentinvention, and the supplementary active compound will have complementaryactivities, that do not adversely affect each other. In a preferredembodiment, the further therapeutic agent is an antagonist offibroblast-growth factor (FGF), hepatocyte growth factor (HGF), tissuefactor (TF), protein C, protein S, platrelet-derived growth factor(PDGF), or HER2 receptor.

The compositions of the invention may be in a variety of forms. Theseinclude for example liquid, semi-solid, and solid dosage forms, but thepreferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions. The preferred mode ofadministration is parenteral (e.g. intravenous, intramuscular,intraperinoneal, subcutaneous). In a preferred embodiment, thecompositions of the invention are administered intravenously as a bolusor by continuous infusion over a period of time. In another preferredembodiment, they are injected by intramuscular, subcutaneous,intra-articular, intrasynovial, intratumoral, peritumoral,intralesional, or perilesional routes, to exert local as well assystemic therapeutic effects.

Sterile compositions for parenteral administration can be prepared byincorporating the antibody, antibody fragment or antibody conjugate ofthe present invention in the required amount in the appropriate solvent,followed by sterilization by microfiltration. As solvent or vehicle,there may be used water, saline, phosphate buffered saline, dextrose,glycerol, ethanol, and the like, as well as a combination thereof. Inmany cases, it will be preferable to include isotonic agents, such assugars, polyalcohols, or sodium chloride in the composition. Thesecompositions may also contain adjuvants, in particular wetting,isotonizing, emulsifying, dispersing and stabilizing agents. Sterilecompositions for parenteral administration may also be prepared in theform of sterile solid compositions which may be dissolved at the time ofuse in sterile water or any other injectable sterile medium.

The antibody, antibody fragment or antibody conjugate of the presentinvention may also be orally administered. As solid compositions fororal administration, tablets, pills, powders (gelatine capsules,sachets) or granules may be used. In these compositions, the activeingredient according to the invention is mixed with one or more inertdiluents, such as starch, cellulose, sucrose, lactose or silica, underan argon stream. These compositions may also comprise substances otherthan diluents, for example one or more lubricants such as magnesiumstearate or talc, a coloring, a coating (sugar-coated tablet) or aglaze.

As liquid compositions for oral administration, there may be usedpharmaceutically acceptable solutions, suspensions, emulsions, syrupsand elixirs containing inert diluents such as water, ethanol, glycerol,vegetable oils or paraffin oil. These compositions may comprisesubstances other than diluents, for example wetting, sweetening,thickening, flavoring or stabilizing products.

The doses depend on the desired effect, the duration of the treatmentand the route of administration used; they are generally between 5 mgand 1000 mg per day orally for an adult with unit doses ranging from 1mg to 250 mg of active substance.

In general, the doctor will determine the appropriate dosage dependingon the age, weight and any other factors specific to the subject to betreated.

Therapeutic Methods of Use

In another embodiment, the present invention provides a method forinhibiting the EphA2 receptor activity by administering an antibodywhich antagonizes said EphA2 receptor, to a patient in need thereof. Anyof the type of antibodies, antibody fragments, or cytotoxic conjugatesof the invention, may be used therapeutically. The invention thusincludes the use of antagonistic anti-EphA2 antibodies, fragmentsthereof, or cytotoxic conjugates thereof as medicaments.

In a preferred embodiment, antibodies, antibody fragments, or cytotoxicconjugates of the invention are used for the treatment of ahyperproliferative disorder in a mammal. In a more preferred embodiment,one of the pharmaceutical compositions disclosed above, and whichcontains an antibody, antibody fragment, or cytotoxic conjugate of theinvention, is used for the treatment of a hyperproliferative disorder ina mammal. In one embodiment, the disorder is a cancer. In particular,the cancer is a metastatic cancer. The antibodies, antibody fragments,and cytotoxic conjugates of the invention can also be used to treat theneovascularization of said cancer tumor.

Accordingly, the pharmaceutical compositions of the invention are usefulin the treatment or prevention of a variety of cancers, including (butnot limited to) the following: carcinoma, including that of the bladder,breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix,thyroid and skin; including squamous cell carcinoma; hematopoietictumors of lymphoid lineage, including leukemia, acute lymphocyticleukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-celllymphoma, Burkitt's lymphoma; hematopoietic tumors of myeloid lineage,including acute and chronic myelogenous leukemias and promyelocyticleukemia; tumors of mesenchymal origin, including fibrosarcoma andrhabdomyoscarcoma; other tumors, including melanoma, seminoma,tetratocarcinoma, neuroblastoma and glioma; tumors of the central andperipheral nervous system, including astrocytoma, neuroblastoma, glioma,and schwannomas; tumors of mesenchymal origin, including fibrosarcoma,rhabdomyoscarama, and osteosarcoma; and other tumors, includingmelanoma, xeroderma pigmentosum, keratoactanthoma, seminoma, thyroidfollicular cancer and teratocarcinoma, and other cancers yet to bedetermined in which EphA is expressed predominantly. In a preferredembodiment, the cancer is a cancer of the lung, breast, colon, prostate,kidney, pancreas, uterus, ovary, cervix and lymphatic organs,osteosarcoma, synovial carcinoma, a sarcoma, head and neck, a glioma,gastric, liver or other carcinomas in which EphA is expressed. Inanother embodiment, said pharmaceutical composition relates to otherdisorders such as, for example, autoimmune diseases, such as systemiclupus, rheumatoid arthritis, and multiple sclerosis; graft rejections,such as renal transplant rejection, liver transplant rejection, lungtransplant rejection, cardiac transplant rejection, and bone marrowtransplant rejection; graft versus host disease; viral infections, suchas mV infection, HIV infection, AIDS, etc.; and parasite infections,such as giardiasis, amoebiasis, schistosomiasis, and others asdetermined by one of ordinary skill in the art.

Similarly, the present invention provides a method for inhibiting thegrowth of selected cell populations comprising contacting target cells,or tissue containing target cells, with an effective amount of anantibody, antibody fragment or antibody conjugate of the presentinvention, or an antibody, antibody fragment or a therapeutic agentcomprising a cytotoxic conjugate, either alone or in combination withother cytotoxic or therapeutic agents.

The method for inhibiting the growth of selected cell populations can bepracticed in vitro, in vivo, or ex vivo. As used herein, “inhibitinggrowth” means slowing the growth of a cell, decreasing cell viability,causing the death of a cell, lysing a cell and inducing cell death,whether over a short or long period of time.

Examples of in vitro uses include treatments of autologous bone marrowprior to their transplant into the same patient in order to killdiseased or malignant cells; treatments of bone marrow prior to itstransplantation in order to kill competent T cells and preventgraft-versus-host-disease (GVHD); treatments of cell cultures in orderto kill all cells except for desired variants that do not express thetarget antigen; or to kill variants that express undesired antigen.

The conditions of non-clinical in vitro use are readily determined byone of ordinary skill in the art.

Examples of clinical ex vivo use are to remove tumor cells or lymphoidcells from bone marrow prior to autologous transplantation in cancertreatment or in treatment of autoimmune disease, or to remove T cellsand other lymphoid cells from autologous or allogeneic bone marrow ortissue prior to transplant in order to prevent graft versus host disease(GVHD). Treatment can be carried out as follows. Bone marrow isharvested from the patient or other individual and then incubated inmedium containing serum to which is added the cytotoxic agent of theinvention. Concentrations range from about 10 μM to 1 pM, for about 30minutes to about 48 hours at about 37° C. The exact conditions ofconcentration and time of incubation, i.e., the dose, are readilydetermined by one of ordinary skill in the art. After incubation thebone marrow cells are washed with medium containing serum and returnedto the patient by i.v. infusion according to known methods. Incircumstances where the patient receives other treatment such as acourse of ablative chemotherapy or total-body irradiation between thetime of harvest of the marrow and reinfusion of the treated cells, thetreated marrow cells are stored frozen in liquid nitrogen using standardmedical equipment.

For clinical in vivo use, the antibody, the epitope-binding antibodyfragment, or the cytotoxic conjugate of the invention will be suppliedas solutions that are tested for sterility and for endotoxin levels.Examples of suitable protocols of cytotoxic conjugate administration areas follows. Conjugates are given weekly for 4 weeks as an i.v. boluseach week. Bolus doses are given in 50 to 100 ml of normal saline towhich 5 to 10 ml of human serum albumin can be added. Dosages will be 10μg to 100 mg per administration, i.v. (range of 100 ng to 1 mg/kg perday). More preferably, dosages will range from 50 μg to 30 mg. Mostpreferably, dosages will range from 1 mg to 20 mg. After four weeks oftreatment, the patient can continue to receive treatment on a weeklybasis. Specific clinical protocols with regard to route ofadministration, excipients, diluents, dosages, times, etc., can bedetermined by one of ordinary skill in the art as the clinical situationwarrants.

Diagnostic

The antibodies or antibody fragments of the invention can also be usedto detect EphA2 in a biological sample in vitro or in vivo. In oneembodiment, the anti-EphA2 of the invention are used to determine thelevel of EphA2 in a tissue or in cells derived from the tissue. In apreferred embodiment, the tissue is a diseased tissue. In a preferredembodiment of the method, the tissue is a tumor or a biopsy thereof. Ina preferred embodiment of the method, a tissue or a biopsy thereof isfirst excised from a patient, and the levels of EphA2 in the tissue orbiopsy can then be determined in an immunoassay with the antibodies orantibody fragments of the invention. The tissue or biopsy thereof can befrozen or fixed. The same method can be used to determine otherproperties of the EphA2 protein, such as its level of tyrosinephosphorylation, cell surface levels, or cellular localization.

The above-described method can be used to diagnose a cancer in a subjectknown to or suspected to have a cancer, wherein the level of EphA2measured in said patient is compared with that of a normal referencesubject or standard. Said method can then be used to determine whether atumor expresses EphA2, which may suggest that the tumor will respondwell to treatment with the antibodies, antibody fragments or antibodyconjugates of the present invention. Preferrably, the tumor is a cancerof the lung, breast, colon, prostate, kidney, pancreas, uterus, ovary,cervix and lymphatic organs, osteosarcoma, synovial carcinoma, asarcoma, a glioma, gastric, liver, head and neck or other carcinomas inwhich EphA2 is expressed, and other cancers yet to be determined inwhich EphA2 is expressed predominantly.

The present invention further provides for monoclonal antibodies,humanized antibodies and epitope-binding fragments thereof that arefurther labeled for use in research or diagnostic applications. Inpreferred embodiments, the label is a radiolabel, a fluorophore, achromophore, an imaging agent or a metal ion.

A method for diagnosis is also provided in which said labeled antibodiesor epitope-binding fragments thereof are administered to a subjectsuspected of having a cancer, and the distribution of the label withinthe body of the subject is measured or monitored.

Kit

The present invention also includes kits, e.g., comprising a describedcytotoxic conjugate and instructions for the use of the cytotoxicconjugate for killing of particular cell types. The instructions mayinclude directions for using the cytotoxic conjugates in vitro, in vivoor ex vivo.

Typically, the kit will have a compartment containing the cytotoxicconjugate. The cytotoxic conjugate may be in a lyophilized form, liquidform, or other form amendable to being included in a kit. The kit mayalso contain additional elements needed to practice the method describedon the instructions in the kit, such a sterilized solution forreconstituting a lyophilized powder, additional agents for combiningwith the cytotoxic conjugate prior to administering to a patient, andtools that aid in administering the conjugate to a patient.

EXAMPLES Example 1 Generation of Anti-EphA2 Monoclonal AntibodyHybridomas

Four BALB/c VAF mice were immunized with human EphA2-transfected 300-19cells, a pre-B cell line derived from a BALB/c mouse. The stablytransfected cells over-expressing the antigen were generated bytransfection of 300-19 cells with the full-length human EphA2 cDNA andselected for the high expression clones by flow cytometry. Clone 4-6, aclone that highly expresses the human EphA2 receptor on the cellsurface, was selected as immunogen for immunization of mice and forantibody screening of hybridomas. The EphA2-transfected cells weremaintained in the selection medium containing G418 at a finalconcentration of 1 mg/mL and were tested for the EphA2 expressionregularly using a commercially available antibody.

The Balb/c mice were subcutaneously injected with approximately 5×10⁶EphA2-transfected 300-19 cells in 200 μL of phosphate buffered saline(PBS) per mouse. The injections are performed every 2-3 weeks bystandard immunization protocols used at ImmunoGen, Inc. Three daysbefore cell fusion, the mice were intraperitoneally boosted one moretime with the same dose of antigen, and sacrificed for the preparationof spleen cells according to the standard protocols for animal useprocedures on the day of cell fusion.

The spleen was collected from the immunized mouse under sterilizedsurgical conditions and was ground between two sterile and frostedmicroscopic slides to obtain single cell suspension in RPMI-1640 medium.The splenocytes were pelleted and washed twice with RPMI-1640 mediumbefore cell fusion. The spleen cells were mixed and fused with murinemyeloma P3X63Ag8.653 cells (Kearney, J. F. et al., 1979. J. Immunol.,123: 1548-1550) using polyethylene glycol-1500 as fusogen (Roche 783641). After cell fusion and centrifugation, the cells were suspended incomplete RPMI-1640 medium (200 mL) containinghypoxanthine-aminopterin-thymidine (HAT) supplement (Sigma H-0262), andwere plated into ten 96-well flat-bottomed plates (Corning-Costar 3596,200 μL of cell suspension per well). Following incubation at 37° C., 5%CO₂ for 5 days, 100 μL of culture supernatant were removed from eachwell of the plates and replaced with an equal volume of completeRPMI-1640 medium containing hypoxanthine-thymidine (HT) supplement(Sigma H-0137). The incubation (in an atmosphere of 5% CO₂ at 37° C.)was continued until hybridoma clones had grown large enough colonies forantibody screening.

On day 10 post-fusion when hybridoma cells had grown to half confluencein the wells and the supernatant had changed to an orange color,hybridoma supernatants were sampled from the fusion plates for antibodyscreening by immunoassays. For preliminary screening, hybridomasupernatants were tested on EphA2-transfected cells vs. the parental300-19 cells by flow cytometry. Cells were stained with 50 μL ofhybridoma supernatant, followed by incubation with fluorescein-goatanti-mouse IgG (H+L) conjugate, and analyzed by flow cytometry withBecton Dickinson FACSCalibur or FACSArray machine. Hybridoma clones thatanalyzed positive for EphA2-transfected cells but negative for 300-19cells were selected, expanded, frozen for storage, or subcloned bylimiting dilutions to attain a monoclonal population. The specificantibodies secreted by hybridoma cells were isotyped using commerciallyavailable isotyping reagents (Roche 1493027).

Based on flow cytometric data, 29 hybridoma clones, which werespecifically reactive with human EphA2-transfected cells but not withthe parental 300-19 cells, were identified and selected from theimmunization of mice with human EphA2 antigens.

Example 2 Binding Characterization of Anti-EphA2 Antibodies, 37.3D7,37.1F5, 53.2H11, EphA2-N1 and EphA2-N2

The specific binding of each of the purified anti-EphA2 antibodies,37.3D7, 37.1F5 and 53.2H11 was demonstrated by fluorescence activatedcell sorting (FACS) using cells overexpressing human EphA2 and by usingcells that do not express EphA2 (FIGS. 1A, B, and C). Incubation of37.3D7 antibody, or 37.1F5 antibody or 53.2H11 antibody (60 nM) in 100μl cold FACS buffer (1 mg/mL BSA in Dulbecco's MEM medium) was performedusing cells overexpressing EphA2 and cells that do not express EphA2 ina round-bottom 96-well plate on ice. After 1 h, the cells were pelletedby centrifugation and washed with cold FACS buffer and then incubatedwith goat-anti-mouse IgG-antibody-FITC conjugate (100 μL, 6 μg/ml inFACS buffer) on ice for 1 h. The cells were then pelleted, washed, andresuspended in 200 μL of 1% formaldehyde solution in PBS. The cellsamples were then analyzed using a FACSCalibur reader (BD Biosciences).

A strong fluorescence shift was obtained upon incubation of humanEphA2-overexpressing cells with 37.3D7, 37.1F5, or 53.2H11 antibody, incontrast to an insignificant shift upon incubation of cells that do notexpress human EphA2 with 37.3D7, 37.1F5, or 53.2H11 antibody (FIGS. 1A,1B, and 1C), which demonstrates that the 37.3D7, 37.1F5 and 53.2H11antibodies were selectively binding to human EphA2. The positive controlanti-EphA2 antibody, B2D6 (Upstate), showed a similar fluorescence shiftupon incubations with cells that over-expressed human EphA2 (FIG. 1A). Astrong fluorescence shift was also observed by FACS assay using 37.3D7and human cancer cells, such as human breast cancer MDA-MB-231 cells,human colon cancer HT-29 cells, human pancreatic cancer BxPC3 cells,which shows that 37.3D7 antibody binds to human EphA2 on the surface ofhuman tumor cells (FIG. 2). Similar data were also obtained using 37.1F5and 53.2H11 antibodies with human tumor cell lines.

The apparent dissociation constants (K_(D)) for the binding of 37.3D7,37.1F5 and 53.2H11 antibodies with human EphA2 on the surface of cellswas determined by FACS assays of the binding of antibody at severalconcentrations to cells over expressing human EphA2 and human breastcancer MDA-MB-231 cells (FIGS. 3A-3C). The values of K_(D) wereestimated by non-liner regression for one-site binding. The bindingcurves yielded the apparent K_(D) values of 0.3 nM for 37.3D7 antibody,0.07 nM for 37.1F5 antibody, and 0.14 nM for 53.2H11 antibody (FIGS. 3A,3B, and 3C).

Using the same experimental protocol, apparent kD values of 0.18 nM and0.05 nM were determined for EphA2-N1 and EphA2-N2, respectively.

A strong fluorescence shift was obtained upon incubation of cells thatover-express murine EphA2 or rat EphA2 with 37.3D7 antibody or 53.2H11antibody, in contrast to an insignificant shift upon incubation of cellsthat do not express murine EphA2 or rat EphA2 with 37.3D7 antibody or53.2H11 antibody (FIG. 4), which demonstrates that the 37.3D7 and53.2H11 antibodies bind also to murine EphA2 and rat EphA2. A strongfluorescence shift was also observed by FACS assay using 37.3D7 or37.1F5 or 53.2H11 antibody with monkey (Cercopithecus aethiops)epithelial VERO cells (FIG. 5A), which shows that 37.3D7, 37.1F5 and53.2H11 bind to monkey EphA2 as well. The apparent values of K_(D) wereestimated by non-liner regression for one-site binding. The bindingcurves by FACS assay yielded K_(D) values of 0.15 nM for 37.3D7, 0.05 nMfor 37.1F5, and 0.07 nM for 53.2H11 on monkey cells (FIGS. 5B, 5D and5F).

Example 3 Inhibition of Binding of EphrinA1 to MDA-MB-231 cells by37.3D7, 37.1F5, 53.2H11, EphA2-N1 and EphA2-N2 Antibodies

The binding of ephrinA1 to MDA-MB-231 human breast cancer cells wasinhibited by 37.3D7, 37.1F5 and 53.2H11 antibodies (FIG. 6). MDA-MB-231cells were incubated with or without 5 μg/mL 37.3D7, 37.1F5, or 53.2H11antibody for 2 h, followed by incubation with 100 ng/mL biotinylatedephrinA1 for 30 min at 4° C. The cells were then washed twice withserum-free medium to remove unbound biotin-ephrinA1, and were then lysedin 50 mM HEPES buffer, pH 7.4, containing 1% NP-40 and proteaseinhibitors. An Immulon-2HB ELISA plates were coated with a mousemonoclonal anti-EphA2 antibody (D7, Upstate) and were used to capturethe EphA2 and bound biotin-ephrinA1 from the lysate. The binding of thecoated antibody to the cytoplasmic C-terminal domain of the EphA2 didnot interfere with the binding of biotin-ephrinA1 to the extracellulardomain of EphA2. The wells were washed, incubated withstreptavidin-horseradish peroxidase conjugate, washed again, and thendeveloped with ABTS/H₂O₂ substrate. The inhibition of ephrinA1 bindingto MDA-MB-231 cells by 5 μg/mL 37.3D7, 37.1F5, or 53.2H1 antibody wasessentially quantitative; the signal was almost equivalent to that ofthe ELISA background signal obtained using a control lackingbiotin-ephrinA1 (FIGS. 6A, 6B and 6C).

Both EphA2-N1 and Epha2-N2 were capable of inhibiting binding of humanephrinA1 to MDA-MB-231 cells to the same extent as 37.3D7.

Example 4 Inhibition of EphA2 Mediated Cell Signaling by 37.3D7, 37.1F5,53.2H11, EphA2-N1 and EphA2-N2 Antibodies

Treatment of MDA-MB-231 human breast cancer cells with 37.3D7, or 37.1F5antibody completely inhibited intracellular EphA2 receptor signaling asshown by the inhibition of EphA2 receptor autophosphorylation (FIG. 7A)and by the inhibition of phosphorylation of its downstream effectorssuch as Akt (FIG. 7B). Treatment of pancreatic cancer CFPAC-1 cells with37.3D7 antibody or 53.2H11 antibody completely inhibited intracellularEphA2 receptor signaling as shown by the inhibition of EphA2 receptorautophosphorylation (FIG. 7C).

In FIGS. 7A and 7C, the mammary MDA-MB-231 cells or the pancreaticCFPAC-1 cells were grown in regular medium (as suggested from ATCC foreach cell line) with serum for 3 days, then cultured in serum-freemedium for 12-14 h. Serum-starved cells were treated with 15 μg/mL37.3D7, 37.1F5, or 53.2H11 antibody or control IgG₁ for 2 h, followed bystimulation with 1 μg/mL ephrin A1-Fc (R&D) for 10 min at 37° C. Thecells were then lysed in ice-cold lysis buffer containing protease andphosphatase inhibitors (50 mM HEPES buffer, pH 7.4, 1% NP-40, 1 mMsodium orthovanadate, 100 mM sodium fluoride, 10 mM sodiumpyrophosphate, 2.5 mM EDTA, 10 μM leupeptin, 5 μM pepstatin, 1 mM PMSF,5 mM benzamidine, and 5 μg/mL aprotinin). The lysates wereimmunoprecipitated with anti-EphA2 antibody D7 (Upstate) coupled toprotein A/G beads. The immunoprecipitated EphA2 was resolved on anSDS-polyacrylamide gel and Western blotted with phosphotyrosine specificmonoclonal antibody, 4G10 (Cell Signaling Technology). To evaluate theEphA2 protein level in each immunoprecipated sample, the same membranewas re-blotted with anti-EphA2 antibody, D7 (Upstate). Use of a controlantibody showed no inhibition of the ephrin A1-stimulatedautophosphorylation of EphA2 receptor (FIG. 7C). In contrast, a completeinhibition of the ephrinA1-stimulated autophosphorylation of EphA2receptor was obtained upon treatment with 37.3D7, 37.1F5, or 53.2H11antibody (FIGS. 7A and 7C). The ephrin A1-stimulated activation of thedownstream effectors, such as Akt, was also inhibited in MDA-MB-231cells by 37.3D7 or 37.1F5 antibody, as shown using Western blots oflysates and rabbit polyclonal anti-phospho-Ser⁴⁷³ Akt antibody (CellSignaling Technology) (FIG. 7B).

The 37.3D7 and 53.2H11 antibodies by themselves did not stimulate EphA2autophosphorylation in human breast cancer MDA-MB-231 cells, in contrastto the stimulatory effect of ephrinA1 on EphA2 autophosphorylation inMDA-MB-231 cells (FIGS. 8A and 8B). Similar data were obtained for the37.1F5 antibody using MDA-MB-231 cells. In FIG. 8, the MDA-MB-231 cellswere grown in regular medium with serum for 3 days, then cultured inserum-free medium for 12-14 h. Serum-starved cells were treated with 1μg/mL ephrinA1-Fc or 15 μg/mL 37.3D7 or 53.2H11 antibody for 10 min. Thecell lysates were subjected to immunoprecipitation with anti-EphA2antibody, D7 (Upstate). After separation on a SDS-polyacrylamide gel,the blot was probed with anti-phosphotyrosine antibody, 4G10 (CellSignaling Technology) and anti-EphA2 antibody D7 (Upstate). Similarresults were obtained with both EphA2-N1 and EphA2-N2 in human breastcancer MDA-MB-231 cells, as neither antibody stimulates EphA2autophosphorylation by itself, whereas each of them preventsephrinA1-dependent phosphorylation of the EphA2 receptor.

The 37.3D7, 37.1F5, 53.2H11, EphA2-N1, and EphA2-N2 antibodies aretherefore unique among all known anti-EphA2 antibodies in theireffectiveness to inhibit ephrinA1-stimulated EphA2 intracellularsignaling.

Example 5 Inhibition of Serum-Stimulated Growth and Survival of HumanTumor Cells by 37.3D7 and 53.2H11 Antibodies

Several human tumor cell lines were tested in serum-free conditions fortheir growth and survival response to serum in the presence of 37.3D7 or53.2H11 antibody. Approximately 3000 cells/well were plated in a 96-wellplate in regular medium (as suggested from ATCC for each cell line) withserum, which was replaced with serum-free medium the following day.

After one day of growth in serum-free medium, the cells were incubatedwith 15 μg/mL 37.3D7 antibody or 53.2H11 antibody or control IgG₁followed by the addition of serum to obtain a final concentration of 1%or 1.5% serum. The cells were then allowed to grow for another 3 days. Asolution of MTT [3-(4,5)-dimethylthiazol-2-yl-2,3-diphenyltetrazoliumbromide; 25 μL of a 5 mg/mL solution in PBS] was then added and thecells were returned to the incubator for 2-3 h. The medium was thenremoved and replaced by 100 μL DMSO, mixed, and the absorbance of theplate was measured at 545 nm. Several human tumor cell lines showed agrowth and survival response upon addition of serum that wassignificantly inhibited by 37.3D7 or 53.2H11 antibody. As examples, thefindings with the colon tumor cell lines, HT-29, LoVo; the pancreatictumor cell line, CFPAC-2, BxPC3; and melanoma UACC-257 are shown.

The 37.3D7 antibody strongly inhibited serum-stimulated growth andsurvival of human colon cancer HT-29 cells (FIG. 9A). In anotherexperiment, the 37.3D7 antibody strongly inhibited serum-stimulatedgrowth and survival of BxPC3 human pancreatic cancer cells in adose-dependent manner with an IC₅₀ value of 4 nM (FIG. 10A). Inaddition, the 37.3D7 or 53.2H11 antibody strongly inhibitedserum-stimulated growth and survival of LoVo human colon cancer cells(FIG. 9B), CFPAC-1 human pancreatic cancer cells (FIG. 9C) and UACC-257melanoma cancer cells (FIG. 9D) and the 53.2H11 antibody inhibited serumor EGF-stimulated growth and survival of LoVo cells in a dose-dependentmanner with an IC₅₀ value of 2 nM (FIGS. 10B and 10C). In FIG. 10, OD₅₄₅values for 0% serum-treated samples were set to 100% inhibition and 0%inhibition was set using samples treated with 1.5% serum or 10 ng/mlEGF. None of the previous reported anti-EphA2 antibodies have inhibitoryactivities on the anchorage-dependent (monolayer) growth of human tumorcells. Therefore, 37.3D7 and 53.2H11 antibodies are unique in theirability to inhibit anchorage-dependent growth (monolayer growth) ofhuman tumor cells.

Example 6 Inhibition of VEGF-Mediated Cell Signaling and VEGF-StimulatedGrowth and Survival of Human Umbilical Vein Endothelial Cells (HUVECs)by 37.3D7, 37.1F5, and 53.2H11 Antibody

A strong fluorescence shift was obtained upon incubation of HUVEC cellswith 37.3D7, 37.1F5, or 53.2H11 antibody by FACS analysis, indicatingthat 37.3D7, 37.1F5 and 53.2H11 antibodies bind to EphA2 receptorsexpressed on HUVEC cells. The apparent dissociation constants (K_(D))for the binding of 37.3D7, 37.1F5, and 53.2H11 antibodies with EphA2 onthe surface of the cells were determined from the binding curvesestablished with FACS binding assays performed at several concentrationsand shown in FIG. 11. A value of K_(D)=0.3 nM for the 37.3D7 antibodywas estimated by non-liner regression for one-site binding (FIG. 11),which is similar to the K_(D) value of the binding of 37.3D7 antibody tohuman cancer cells. A value of K_(D)=0.01 nM for the 37.1F5 antibody anda value of K_(D)=0.06 nM for the 53.2H11 antibody were similarlyobtained. This indicates that 37.3D7, 37.1F5 and 53.2H11 antibodiesspecifically bind to HUVEC cells through the EphA2 receptor.

The 37.3D7 antibody strongly inhibited VEGF-induced HUVEC growth andsurvival. The activity is similar or better than that of Avastin®, ananti-VEGF blocking antibody (Genentech) (FIG. 12). An agonisticanti-EphA2 antibody did not inhibit VEGF-induced HUVEC growth andsurvival (FIG. 12). In FIG. 12, HUVEC cells were grown in EBM-2 mediumwith serum and endothelial cell (EC) supplements (Clonetics) for 3 days.Cells were cultured in serum-free medium plus EC growth supplementslacking VEGF for 12-14 h. Following serum starvation, cells werestimulated with 5 ng/mL VEGF plus 0.4% serum with or without indicatedantibodies (100 μg/mL). The effects of the antibodies on VEGF-inducedHUVEC cell growth and survival was determined 3 days after addition ofantibodies and VEGF using the MTT assay as described in Example 5. Thepercent inhibition of VEGF-mediated growth and survival by antibodies isshown in FIG. 12. OD₅₄₅ values for vehicle-treated samples were set to0% inhibition and 100% inhibition was set using samples lacking VEGF.

That treatment of HUVEC cells with 37.3D7 antibody inhibitsintracellular EphA2 receptor signaling was shown by measuring theinhibition of phosphorylation of its downstream effectors such as Akt.

The inhibition is similar to that of Avastin®, an anti-VEGF blockingantibody (Genentech) (FIG. 13). agonistic anti-EphA2 antibody did notinhibit VEGF-induced Akt phosphorylation in HUVEC cells. In FIG. 13,HUVEC cell were starved for 12-14 h in serum-free medium plus ECsupplements lacking VEGF. Cells were treated with antibodies (20 μg/mL)for 1 h before addition of VEGF (100 ng/mL). Cells were lysed 15 minafter VEGF addition and immunoblots were probed with the indicatedantibodies.

Example 7 Suppression of Growth of Human Colon Cancer HT-29 Xenograft inMice by 37.3D7 Antibody (FIG. 14)

Human colon cancer HT-29 xenografts were established in SCID mice bysubcutaneous injection of 2×10⁶ HT-29 cells. When the mice showedpalpable (50 mm³) HT-29 xenograft tumors, they were treated with 37.3D7antibody or a control antibody (IgG₁) (1 mg/mouse, i. v., two times perweek) or PBS alone (100 μL/mouse, i. v., two times per week). The growthof tumors was significantly slowed by 37.3D7 antibody treatment comparedto a control antibody treatment or PBS alone. No toxicity of 37.3D7antibody was observed, based on measurements of the weights of the mice.

Example 8 Inhibition of Early Mammary MDA-MB-231 Metastasis by theAnti-EphA2 Antibody hu53.2H11

Anti-tumor activity of the anti-EphA2 antibody hu532H11 was evaluated atone dose level against early mammary MDA-MB-231 tumor implantedsubcutaneously in female SCID mice. The effect of this antibody on theMDA-MB-231 tumor invasion in the superficial axial and inguinal lymphnodes was also investigated. To do so, hu532H11 was administered at 40mg/kg/adm by iv route, on days 1, 5, 8, 12, 15, 19, 22 and 26 post tumorimplantation. Control group was left untreated.

For the evaluation of anti-tumor activity of hu532H11, animals wereweighed daily and tumors were measured 2-3 times weekly by caliper.Tumor weights were calculated using the formula mass (mg)=[length(mm)×width (mm)²]/2. Antitumor activity was evaluated according to 3criteria: 1) including T/C, defined as median tumor weight (mg) of atreated group divided by median tumor weight of the nontreated control;2) the determination of tumor growth delay (T-C), where T is defined asthe median time in days required for treatment group tumors to reach 750mg and C is the median time for the control group tumors to reach thesame size, and 3) tumor cell kill is defined as log 10 cell kill(gross)=[T−C value in days]/(Td×3.32). T-C is defined above, and Td isthe tumor volume doubling time in days of the control tumors, which isestimated from the best fit straight line from a log-linear growth plotof the control group tumors in exponential growth (100-1,000 mg range).

In a parallel study, animals were treated as described before, and onday 28 post tumor implantation all mice were sacrificed and axillary andinguinal lymph nodes were collected (median tumor size in the controlgroup=1558 mg). The human Ki67 antibody was used in order tospecifically identify MDA-MB-231 tumor cells in the lymph nodes byimmunostaining. The surface area of metastases in lymph nodes wascalculated (mean of 2 sections) as S=human Ki67 surface area×100/lymphnode surface area.

Efficacy on Primary Tumor:

-   -   hu532H11 was well tolerated at 40 mg/kg/adm (total dose 320        mg/kg) with +8.9% body weight change on day 27. This dose        delayed tumor growth of the primary tumor (T/C=27% and 1.0 log        cell kill gross), even though the tumor escaped under therapy.

Anti-Metastatic Activity:

-   -   hu532H11 induced a reduction of metastases surface (>50%) in        both axilliary and inguinal lymph nodes.

In conclusion in mice bearing mammary tumor MDA-MB-231, hu532H11 delaysthe growth of the primary tumor treated at an early stage of tumordevelopment (T/C=27% and 1.0 log cell kill gross), and reduces themetastases surface (>50%) in both axillary and inguinal lymph nodes.

In another study, the activity of an anti-EphA2 antibody can beevaluated in the human colon HT29 liver “metastasis” model. The murineanti-EphA2 antibody 53.2H11 is administered iv, twice weekly, from day 4post intrasplenic implantation of HT29 cells in SCID female mice (n=20mice per group for non-tumor bearing animals (NTBA), treated andcontrol). On day 50, 3 days post the 13^(th) anti-EphA2 administration,the mice are necropsied and their spleen and liver are weighed in orderto evaluate tumor mass either at the primary tumor site (spleen) or atsite of metastasis (liver). Number of metastases is also evaluated. Datais analysed using the statistical tools known to the person skilled inthe art.

-   -   Treatment with anti-EphA2 at 40 mg/kg/inj (total dose of 520        mg/kg) is well tolerated.    -   Primary tumor weight (spleen): A significant difference of        spleen weight is observed between NTBA and control-implanted        mice, the latter being bigger. There is no significant        difference between spleen weight of control implanted mice and        the one of anti-EphA2 treated mice.    -   Metastases weight (liver): the liver weight of control-implanted        mice is significantly bigger than the liver weight of NTBA; on        the other hand, it is significantly smaller in anti-EphA2        treated mice than in control-implanted mice.    -   Number of liver metastases: no statistical difference is        observed between control implanted and anti-EphA2 treated mice.

In conclusion, intrasplenic implantation of human colic adenocarcinomaHT-29 significantly induces an increase in liver weight due to themetastatic tumor burden. Anti-EphA2 treatment is able to significantlydecrease metastatic tumor burden as observed by the reduction of theliver weight of implanted mice without affecting the number ofmetastases counted on the liver.

Example 9 Cloning and Sequencing of the Light and Heavy Chains of 37.1F5Antibody

RNA Preparation from Hybridoma Cells that Produces the 37.1F5 Antibody

Preparations of total RNA were obtained from 5×10⁶ hybridoma cells,which produce 37.1F5 antibody, using Qiagen's RNeasy miniprep kit.Briefly, 5×10⁶ cells were pelleted and resuspended in 350 μL RLT buffer(containing 1% β-mercaptoethanol). The suspension was homogenized bypassing it through a 21.5 gauge needle and syringe roughly 10-20 timesor until it was no longer viscous. Ethanol (350 μL of 70% aqueousethanol) was added to the homogenate, which was mixed well. The solutionwas transferred to a spin column, placed in a 2-mL collection tube andspun at >8000×g for 15 seconds. The column was washed twice with 500 μLRPE buffer, then transferred to a fresh tube and eluted with 30 μL RNasefree water and a 1-minute spin. The eluate (30 μL) was placed back onthe column for a second 1-minute elution spin. An aliquot of the 30 μLeluate was diluted with water and used to measure the UV absorption at260 nm for RNA quantitation.

cDNA Preparation with Reverse Transcriptase (RT) Reaction

The variable region 37.1F5 antibody cDNA was generated from the totalRNA using Invitrogen's SuperscriptII kit. The kit protocols werefollowed closely, utilizing up to 5 μg of total RNA from the Qianeasymini preps. Briefly, the RNA, 1 μL random primers, and 1 μL dNTP mixwere brought up to 12 μL with RNase free sterile distilled water andincubated at 65° C. for 5 minutes. The mix was then put on ice for atleast 1 minute. Next 4 μL of 5×reaction buffer, 2 μL 0.1 M DTT, and 1 μLRNaseOUT were added and the mix was incubated at 25° C. for 2 minutes inan MJ Research thermalcycler. The thermalcycler was paused so that 1 μLof SuperscriptII enzyme could be added and then restarted for anadditional 10 minutes at 25° C. before shifting to 55° C. for 50minutes. The reaction was heat inactivated by heating to 70° C. for 15min and the RNA was removed by adding 1 μL RNase H and incubating at 37°C. for 20 minutes.

Degenerate PCR Reactions

The procedure for the first round degenerate PCR reaction on the cDNAderived from hybridoma cells was based on methods described in Wang etal. (2000; J Immunol Methods.; 233(1-2):167-77) and Co et al. (1992; JImmunol.; 148(4):1149-54). The primers for this round (Table 2) containrestriction sites to facilitate cloning into the pBluescriptII plasmids.

The PCR reaction components (Table 3) were mixed on ice in thin walledPCR tubes and then transferred to an MJ research thermalcycler preheatedand paused at 94° C. The reactions were performed using a programderived from Wang et al. (2000; J Immunol Methods.; 233(1-2):167-77), asfollows:

Name: Wang45

-   -   1) 94° C. 3:00 min    -   2) 94° C. 0:15 sec    -   3) 45° C. 1:00 min    -   4) 72° C. 2:00 min    -   5) Go to 2 29 times    -   6) 72° C. 6:00 min    -   7) 4° C. for ever    -   8) end

The PCR reaction mixtures were then run on a 1% low melt agarose gel,the 300 to 400 bp bands were excised, purified using Zymo DNA minicolumns, and sent to Agencourt biosciences for sequencing. Therespective 5′ and 3′ PCR primers were used as sequencing primers togenerate the 37.1F5 variable region cDNAs from both directions.

Cloning the 5′ End Sequence

Since the degenerate primers used to clone the 37.1F5 variable regionlight chain and heavy chain cDNA sequences alters the 5′end sequences,additional sequencing efforts were needed to decipher the completesequences. The preliminary cDNA sequence from the methods describedabove were used to search the NCBI IgBlast site(http://www.ncbi.nlm.nih.gov/igblast/) for the murine germline sequencesfrom which the 37.1F5 sequence is derived. PCR primers were designed(Table 4) to anneal to the leader sequence of the murine antibody sothat a new PCR reaction could yield the complete variable region cDNA,unaltered by the PCR primers. The PCR reactions, band purifications, andsequencing were performed as described above. The germline sequencesfrom which the light chain and heavy chain of mu37.1F5 are likelyderived, are accessible under the Genbank accession numbers MUSIGKVR3and AF303839, respectively.

Peptide Analysis for Sequence Confirmation

The cDNA sequence information for the variable region was combined withthe germline constant region sequence to obtain full length antibodycDNA sequences. The molecular weights of the heavy chain and light chainwere then calculated and compared with the molecular weights obtained byLC/MS analyses of the murine 37.1F5 antibody.

Table 5 gives the calculated mass from the cDNA sequences for 37.1F5 LCand HC together with the values measured by LC/MS. The molecular weightmeasurements are consistent with the cDNA sequences for both the 37.1F5light and heavy chain.

Essentially the same method was used for cloning of the light and heavychains of 37.3D7 and 53.2H11. The Genbank accession numbers of thegermline sequences from which the light chain and of the heavy chain of37.3D7 are likely derived, are respectively MMU231217 and AF303868. For53.2H11, they are respectively MMU231196 and AF303833; for EphA2-N1,K02161 and J00488 respectively; and for EphA2-N2, AJ231222 and J00488respectively.

Example 10 Inhibition of the Growth of EphA2 Expressing Tumor Cells byHumanized-37.3D7-SPDB-DM4 and Humanized-53.2H11-SPDB-DM4

Humanized 37.3D7 and humanized 53.2H11 antibodies were conjugated toL-DM4 N^(2′)deacetyl-N^(2′)(4-methyl-4-mercapto-1-oxopentyl)-maytansineusing SPDB (4-[2-pyridyldithio]butanoic acid N-hydroxsuccinimde ester)linker. Briefly, the antibody was modified at 8 mg/mL with 5.5 or 6.5folds molar excess of SPDB for hu53.2H11 and hu37.3D7 respectively. Thereaction was carried out in Buffer A (50 mM KP_(i)/50 mM NaCl/2 mM EDTA,pH 6.5, 95% v/v) with EtOH (5% v/v) for 90 minutes at room temperature.The modified antibody was then purified by SephadexG25 desalting columnwith Buffer A. Next, the modified antibody was reacted with a 1.7-foldmolar excess of DM4 over SPDB linker. The reaction was carried out at2.5 mg/mL antibody in Buffer A (97% v/v) and DMA (dimethylacetamide, 3%v/v) at room temperature for 20 hours. The conjugate was purified bySephadexG25 desalting column with 10 mM Histidine, 130 mM Glycine, 5%sucrose, pH5.5. The drug to antibody ratio was 4.0 for hu37.3D7-SPDB-DM4and 3.1 for hu53.2H11-SPDB-DM4.

The effects of hu37.3D7-SPDB-DM4 and hu53.2H11-SPDB-DM4 on the growth ofEphA2 expressing tumor cells were first tested using the in vitro cellproliferation WST-8((2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,monosodium salt) assay (Catalog# CK04-11, Dojindo Molecular Technogies,Inc). Several human tumor cell lines were tested. Approximately 2000cells/well were plated in a 96-well plate in regular medium (assuggested from ATCC for each cell line) with 10% serum in the presenceof a variety of concentration of hu37.3D7-SPDB-DM4 orhu53.2H11-SPDB-DM4. The cells were then allowed to grow for 5 days. Asolution of WST-8 [20 μL solution] was then added and the cells werereturned to the incubator for 2-3 h. The absorbance of the plate wasmeasured at 450 nm and 650 nm. Two control groups were used in theexperiments. 0% survival is the medium only control. 100% survival isthe cells only control. For data analysis, the A650 nm (referencewavelength) values were first subtracted from the corresponding A450 nmvalues. Then, A450 nm values of each sample were normalized bysubtraction of A450 nm values of the background control (medium only).The survival fractions were calculated by the normalized A450 values ofthe samples divided by the normalized A450 values from the cells onlycontrols (100% survival-0% survival). Log [Ab-DM4] values were plottedon the x-axis and survival fractions were plotted on y-axis.

Hu37.3D7-SPDB-DM4 and hu53.2H11-SPDB-DM4 significantly inhibited thegrowth of EphA2 expressing human tumor cells, including PC3 prostatetumor cells, MDA-MDA-MB-231 breast tumor cells, WM-115 melanoma cells,A375 melanoma cells and LoVo colon tumor cells. As an example, thefindings with the PC3 prostate tumor cells are shown. Thehu37.3D7-SPDB-DM4 or hu53.2H11-SPDB-DM4 strongly inhibited the growth ofPC3 cells in a dose-dependent manner with an similar IC₅₀ value of 0.02nM (FIGS. 15A & 15B). The potency of conjugates correlated with theEphA2 expression levels. More than 50-fold higher concentration ofhu37.3D7-SPDB-DM4 or hu53.2H11-SPDB-DM4 was required to inhibit thegrowth of the SK-MeI28 cells (IC50 values: 1.3 nM and >5 nM,respectively; FIGS. 15A & 15B), which expressed almost undetectablelevel of EphA2 on cell surface (measured by FACS data not shown) (FIGS.15A & 15B). Therefore, results of the in vitro growth inhibition assaysdemonstrated the ability of antagonist anti-EphA2 antibody-conjugates tospecifically inhibit the growth of EphA2 expressing tumor cell lines.

The effects of hu37.3D7-SPDB-DM4 and hu53.2H11-SPDB-DM4 on the growth ofEphA2 expressing tumor xenografts were tested. A study using MDA-MB-231breast tumor xenograft model is shown as one example. Human breastcancer MDA-MB-231 xenografts were established in female CB17 SCID mice 5weeks of age by subcutaneous injection of 1×10⁷ MDA-MB-231 cells. WhenMDA-MB-231 xenograft tumors were established (average size of 83 mm³),mice were treated with a single i. v injection of hu3D7-SPDB-DM4 orhu2H11-SPDB-DM4 or PBS. The doses of antibodies were 15 mg/kg of mousebody weight, 7.5 mg/kg of mouse body weight and 3.25 mg/kg of mouse bodyweight. The growths of MDA-MB-231 tumors were completely inhibited byeither hu3D7-SPDB-DM4 or hu2H11-SPDB-DM4 antibody-conjugates at all ofthe tested concentrations except at 3.25 mg/kg of hu2H11-SPDB-DM4, whichshows marked delay of tumor cell growth relative to PBS control (FIGS.16A & 16B). The median tumor volumes in each group (6 mice per group)are shown in the FIGS. 16A & B. In summary, both hu3D7-SPDB-DM4 andhu2H11-SPDB-DM4 have potent growth inhibitory activities on EphA2expressing tumors in vivo. No toxicities of both antibody-conjugateswere observed, based on the body weight measurements.

TABLES

TABLE 1A The mu37.3D7 light chain framework surface residues andcorresponding residues at the same Kabat position in the human 28E4antibody. The residues that are different and therefore changed in thehu37.3D7 antibody are in grayed boxes. mu37.3D7 Light Chain FrameworkSurface Residues And Corresponding Residues In The Human 28E4 Antibody

TABLE 1B The mu37.3D7 heavy chain framework surface residues andcorresponding residues at the same Kabat position in the human 28E4antibody. The residues that are different and therefore changed in thehu37.3D7 antibody are in grayed boxes. The starred (*) residues are backmutated to the mu37.3D7 residue in one or more hu37.3D7 variants.mu37.3D7 Heavy Chain Framework Surface Residues And CorrespondingResidues In The Human 28E4 Antibody

TABLE 2 Primers used for the degenerate PCRreactions are based on those in Wanget al., 2000 except HindKL (SEQ ID NO: 58)which is based on Co et al. 1992. Mixed bases are defined as follows: H = A + T + C, S = g + C, Y = C + T, K = G + T, M = A + C, R = A + g,W = A + T, V = A + C + G. Primer Sequence BamIgG1GGAGGATCCATAGACAGATGGGG (SEQ ID NO: 53) GTGTCGTTTTGGC IgG2AbamGGAGGATCCCTTGACCAGGCATC (SEQ ID NO: 54) CTAGAGTCA EcoMH1CTTCCGGAATTCSARGTNMAGCT (SEQ ID NO: 55) GSAGSAGTC EcoMH2CTTCCGGAATTCSARGTNMAGCT (SEQ ID NO: 56) GSAGSAGTCWGG SacIMKGGAGCTCGAYATTGTGMTSACMC (SEQ ID NO: 57) ARWCTMCA HindKLTATAGAGCTCAAGCTTGGATGGT (SEQ ID NO: 58) GGGAAGATGGATACAGTTGGTGC

TABLE 3 The light and heavy chain PCR reaction mixes for cloning of the37.1F5 variable region cDNA sequences. Light Chain Reaction Mix HeavyChain Reaction Mix 5 μl 10 X PCR reaction buffer 5 μl 10 X PCR reactionbuffer (Roche) (Roche) 4 μl 10 mM dNTP mix (2.5 mM 4 μl 10 mM dNTP mix(2.5 mM each) each) 2 μl Template (RT reaction) 2 μl Template (RTreaction) 5 μl 10 μM Sac1MK left primer 2.5 μl 10 μM EcoMH1 left primer5 μl 10 μM HindKL right primer 2.5 μl 10 μM EcoMH2 left primer 5 μl DMSO5 μl 10 μM BamIgG1 right primer 0.5 μl Taq Polymerase (Roche) 5 μl DMSO23.5 μl sterile distilled H₂O 0.5 μl Taq Polymerase (Roche) 23.5 μlsterile distilled H₂O 50 μl Total 50 μl Total

TABLE 4 The 5′end murine leader sequence primersused for the 37.1F5 second round PCRreactions. The 3′end primers are identicalto those used in the first round reactionssince they prime to the respective constant region sequences. PrimerSequence Light Chain GACAGACACACTCCTGCTATGGG 38SB13 LC Leader(SEQ ID NO: 59) Heavy Chain GCAGAATTCATGGGATGGAGCYG 5F85 HC LeaderGATCTTTCT (SEQ ID NO: 60)

TABLE 5 The cDNA calculated and LC/MS measured molecular weights of themurine 37.1 F5 antibody light and heavy chains. Light Chain Heavy ChainDiffer- Differ- cDNA LC/MS ence cDNA LC/MS ence 37.1 F5 24031 Da 24029Da 2 Da 49316 Da 49333 Da 17 Da

1. An antibody or an epitope-binding fragment thereof that specificallybinds to an EphA2 receptor and is an antagonist of said receptor.
 2. Theantibody or epitope-binding fragment thereof according to claim 1,wherein one or more of the following conditions is met: a) said antibodyor epitope-binding fragment thereof is a monoclonal antibody b) saidantibody or epitope-binding fragment thereof is a Fab, Fab′, F(ab′)₂ orF_(v) fragment; c) said antibody or epitope-binding fragment thereof iscapable of inhibiting growth of a cancer cell; d) said antibody orepitope-binding fragment thereof is capable of inhibiting migration of acancer cell; e) said antibody or epitope-binding fragment thereof iscapable of inhibiting angiogenesis; f) said antibody or epitope-bindingfragment thereof is devoid of agonist activity; g) said antibody orepitope-binding fragment thereof is capable of inhibiting the binding ofa ligand to said receptor; h) said antibody or epitope-binding fragmentthereof is capable of inhibiting EphA2 tyrosine phosphorylation; i) saidantibody or epitope-binding fragment thereof is capable of inhibitingEphA2-mediated signaling; j) said antibody or epitope-binding fragmentthereof binds EphA2 with a K_(D) of 3×10⁻¹⁰ M or smaller; and k) saidEphA2 receptor is human. 3-5. (canceled)
 6. The antibody or anepitope-binding fragment thereof according to claim 2, wherein one ormore of the following conditions are met: a) said cancer cell is a cellof a cancer selected from the group consisting of a breast cancer, coloncancer, endometrial cancer, ovarian carcinoma, osteosarcoma, cervicalcancer, prostate cancer, lung cancer, synovial carcinoma pancreaticcancer, a sarcoma, a glioma, head and neck cancer, gastric cancer, livercancer, and other carcinomas; b) said antibody or epitope-bindingfragment thereof does not stimulate EphA2 tyrosine phosphorylation; c)said ligand is ephrinA1; d) said antibody or epitope-binding fragmentthereof is capable of inhibiting EphA2 tyrosine phosphorylation inpresence of ephrinA1; and e) wherein the inhibition of EphA2-mediatedsignaling results in an increase in Akt phosphorylation. 7-17.(canceled)
 18. An antibody or epitope-binding fragment thereof accordingto claim 1, wherein one or more of the following conditions are met: a)said antibody or epitope-binding fragment thereof comprises one or morecomplementarity-determining region having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, and 72; b) said antibody or epitope-binding fragmentthereof comprises a light chain variable region having an amino acidsequence selected from the group consisting of SEQ ID NOs: 26, 28, 30,78, and 80; c) said antibody or epitope-binding fragment thereofcomprises a heavy chain variable region having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 20, 22, 24, 74, and76; d) said antibody or epitope-binding fragment thereof comprises atleast one heavy chain and at least one light chain, wherein said heavychain comprises three sequential complementarity-determining regionshaving amino acid sequences represented by SEQ ID NOs: 1, 2, and 3, andwherein said light chain comprises three sequentialcomplementarity-determining regions having amino acid sequencesrepresented by SEQ ID NOs: 4, 5, and 6; e) said antibody orepitope-binding fragment thereof comprises at least one heavy chain andat least one light chain, wherein said heavy chain comprises threesequential complementarity-determining regions having amino acidsequences represented by SEQ ID NOs: 7, 8, and 9, and wherein said lightchain comprises three sequential complementarity-determining regionshaving amino acid sequences represented by SEQ ID NOs: 10, 11, and 12;f) said antibody or epitope-binding fragment thereof comprises at leastone heavy chain and at least one light chain, wherein said heavy chaincomprises three sequential complementarity-determining regions havingamino acid sequences represented by SEQ ID NOs: 13, 14, and 15, andwherein said light chain comprises three sequentialcomplementarity-determining regions having amino acid sequencesrepresented by SEQ ID NOs: 16, 17, and 18; g) said antibody orepitope-binding fragment thereof comprises at least one heavy chain andat least one light chain, wherein said heavy chain comprises threesequential complementarity determining regions having amino acidsequences represented by SEQ ID NOs: 61, 62, and 63, and wherein saidlight chain comprises three sequential complementarity-determiningregions having amino acid sequences represented by SEQ ID NOs: 64, 65,and 66; h) said antibody or epitope-binding fragment thereof comprisesat least one heavy chain and at least one light chain, wherein saidheavy chain comprises three sequential complementarity determiningregions having amino acid sequences represented by SEQ ID NOs: 67, 68,and 69, and wherein said light chain comprises three sequentialcomplementarity-determining regions having amino acid sequencesrepresented by SEQ ID NOs: 70, 71, and 72; and i) said antibody orepitope-binding fragment thereof is a murine antibody or epitope-bindingfragment thereof and is produced by a hybridoma designated 37.3D7,wherein said hybridoma is deposited at the American Type CultureCollection under the accession number PTA-7660; a hybridoma designated37.1F5, wherein said hybridoma is deposited at the American Type CultureCollection under the accession number PTA-7661; a hybridoma designated53.2H11, wherein said hybridoma is deposited at the American TypeCulture Collection under the accession number PTA-7662; a hybridomaEphA2-N1, wherein said hybridoma is deposited at the American TypeCulture Collection under the accession number PTM-8407; or a hybridomadesignated EphA2-N2, wherein said hybridoma is deposited at the AmericanType Culture Collection under the accession number PTM-8408. 19-36.(canceled)
 37. A humanized or resurfaced antibody or epitope-bindingfragment thereof that binds the same epitope as an antibody orepitope-binding fragment thereof according to claim
 18. 38. A humanizedor resurfaced antibody or epitope-binding fragment thereof according toclaim 37 wherein one or more of the following conditions are met: a)said humanized or resurfaced antibody or epitope-binding fragmentthereof comprises one or more complementarity-determining region havingan amino acid sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, and 72 b) said humanized orresurfaced antibody or epitope-binding fragment thereof comprises alight chain variable region having an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 47, 48, 49, 50, and 52; c) saidhumanized or resurfaced antibody or epitope-binding fragment thereofcomprises a heavy chain variable region having an amino acid sequenceselected from the group consisting of SEQ ID NOS: 32, 34, 36, 37, 38,40, 42, 43, and 45; d) said humanized or resurfaced antibody orepitope-binding fragment thereof comprises at least one heavy chain andat least one light chain, wherein said heavy chain comprises threesequential complementarity-determining regions having amino acidsequences represented by SEQ ID NOS: 1, 2, and 3, and wherein said lightchain comprises three sequential complementarity-determining regionshaving amino acid sequences represented by SEQ ID NOS: 4, 5, and 6; e)said humanized or resurfaced antibody or epitope-binding fragmentthereof comprises at least one heavy chain and at least one light chain,wherein said heavy chain comprises three sequentialcomplementarity-determining regions having amino acid sequences by SEQID NOS: 7, 8, and 9, and wherein said light chain comprises threesequential complementarity-determining regions having amino acidsequences represented by SEQ ID NOS: 10, 11, and 12; f) said humanizedor resurfaced antibody or epitope-binding fragment thereof comprises atleast one heavy chain and at least one light chain, wherein said heavychain comprises three sequential complementarity-determining regionshaving amino acid sequences represented by SEQ ID NOS: 13, 14, and 15,and wherein said light chain comprises three sequentialcomplementarity-determining regions having amino acid sequencesrepresented by SEQ ID NOS:16, 17, and 18; g) said humanized orresurfaced antibody or epitope-binding fragment thereof comprises atleast one heavy chain and at least one light chain, and said heavy chaincomprises three sequential complementarity-determining regions havingamino acid sequences selected from the group consisting of SEQ ID NOs:61, 62, and 63, and said light chain comprises three sequentialcomplementarity-determining regions having amino acid sequences selectedfrom the group consisting of SEQ ID NOs: 64, 65, and 66; h) saidhumanized or resurfaced antibody or epitope-binding fragment thereofcomprises at least one heavy chain and at least one light chain, andsaid heavy chain comprises three sequential complementarity-determiningregions having amino acid sequences selected from the group consistingof SEQ ID NOs: 67, 68, and 69, and said light chain comprises threesequential complementarity-determining regions having amino acidsequences selected from the group consisting of SEQ ID NOs: 70, 71, and72; and i) said humanized or resurfaced antibody or epitope-bindingfragment thereof is selected from a group consisting of hu37.3D7,hu37.1F5, hu53.2H11, huEphA2-N1, and huEphA2-N2. 39-52. (canceled)
 53. Aconjugate comprising the antibody or epitope-binding fragment thereofaccording to claim 1 linked to a cytotoxic agent.
 54. The conjugate ofclaim 53, characterized in that said cytotoxic agent is selected fromthe group consisting of a maytansinoid, a small drug, a tomaymycinderivative, a leptomycin derivative, a prodrug, a taxoid, CC-1065 and aCC-1065 analog.
 55. The conjugate of claim 53, characterized in thatsaid cytotoxic agent is: a) the maytansine DM1 of formula:

b) the maytansine DM4 of formula:

c) a tomaymycin derivative selected group the mu consisting of:8,8′-[1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[5-methoxy-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[1,5-pentanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[1,4-butanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[3-methyl-1,5-pentanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[2,6-pyridinediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[4-(3-tert-butoxycarbonylaminopropyloxy)-2,6-pyridinediylbis-(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[5-(3-aminopropyloxy)-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[5-(N-methyl-3-tert-butoxycarbonylaminopropyl)-1,3-benzenediylbis-(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-{5-[3-(4-methyl-4-methyldisulfanyl-pentanoylamino)propyloxy]-1,3-benzenediylbis(methleneoxy)}-bis[(S)-2-eth-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[5-acetylthiomethyl-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-methylene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]bis-{2-[(S)-2-methylene-7-methoxy-5-oxo-1,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy]-ethyl}-carbamicacid tert-butyl ester8,8′-[3-(2-acetylthioethyl)-1,5-pentanediylbis(oxy)]-bis[(S)-2-methylene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[5-(N-4-mercapto-4,4-dimethylbutanoyl)amino-1,3-benzenediylbis(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[5-(N-4-methyldithio-4,4-dimethylbutanoyl)-amino-1,3-benzenediylbis(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[5-(N-methyl-N-(2-mercapto-2,2-dimethylethyl)amino-1,3-benzenediyl(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[5-(N-methyl-N-(2-methyldithio-2,2-dimethylethyl)amino-1,3-benzenediyl(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(4-(2-(4-mercapto-4-methyl)-pentanamido-ethoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(1-(2-(4-methyl-4-methyldisulfanyl)-pentanamido-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(4-(3-(4-methyl-4-methyldisulfanyl)-pentanamido-propoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(4-(4-(4-methyl-4-methyldisulfanyl)-pentanamido-butoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(4-(3-[4-(4-methyl-4-methyldisulfanyl-pentanoyl)-piperazin-1-yl]-propyl)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(1-(3-[4-(4-methyl-4-methyldisulfanyl-pentanoyl)-piperazin-1-yl]-propyl)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(4-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-ethoxy}-ethoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(1-(2-{2-[2-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(1-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-ethoxy}-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(4-(2-{2-[2-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(1-(2-[methyl-(2-methyl-2-methyldisulfanyl-propyl)-amino]-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(4-(3-[methyl-(4-methyl-4-methyldisulfanyl-pentanoyl)-amino]-propyl)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(4-(3-[methyl-(2-methyl-2-methyldisulfanyl-propyl)-amino]-propyl)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one];and8,8′-[(1-(4-methyl-4-methyldisulfanyl)-pentanamido)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one];or d) a leptomycin derivative selected from the group consisting of:(2-Methylsulfanyl-ethyl)-amid of(2E,10E,12E,16Z,18E)-(R)-6-Hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoicacid (2-methylsulfanyl-ethyl)-amid Bis-[(2-mercaptoethyl)-amid of(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoicacid] (2-Mercapto-ethyl)-amid of(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoicacid (2-Methyldisulfanyl-ethyl)-amid of(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoicacid (2-Methyl-2-methyldisulfanyl-propyl)-amid of(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoicacid; and (2-Mercapto-2-methyl-propyl)-amid of(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoicacid. 56-58. (canceled)
 59. A pharmaceutical composition containing anantibody or epitope-binding fragment thereof according to claim 1 and apharmaceutically acceptable carrier or excipients.
 60. An antibody orepitope-binding fragment thereof according to claim 1 for use as amedicament.
 61. The use of an antibody or epitope-binding fragmentthereof according to claim 1 to make a medicament to treat cancer. 62.The use of claim 61, wherein one or more of the following conditions aremet: a) said cancer is a metastatic cancer; b) said antibody orepitope-binding fragment thereof inhibits tumor neovascularization; andc) said cancer is selected from the group consisting of breast cancer,colon cancer, endometrial cancer, ovarian carcinoma, osteosarcoma,cervical cancer, kidney cancer, prostate cancer, lung cancer, synovialcarcinoma pancreatic cancer, a sarcoma, glioma, head and neck cancer,gastric cancer, liver cancer, and other carcinomas. 63-64. (canceled)65. The use according to claim 61, further comprising the use of afurther therapeutic agent in the manufacture of the same or differentcomposition.
 66. The use according to claim 65 characterized in that thefurther therapeutic agent is an antagonist of fibroblast-growth factor(FGF), hepatocyte growth factor (HGF), tissue factor (TF), protein C,protein S, platelet-derived growth factor (PDGF), or HER2 receptor. 67.A method of diagnosing a cancer in a subject known to or suspected tohave a cancer, said method comprising: a) Contacting cells of saidpatient with an antibody or epitope-binding fragment thereof, b)Measuring the binding of said antibody or epitope-binding fragmentthereof to said cells, and c) Comparing the expression in part (b) withthat of a normal reference subject or standard.
 68. The method of claim67, wherein said cancer is a cell of a cancer selected from the groupconsisting of a breast cancer, colon cancer, endometrial cancer, ovariancarcinoma, osteosarcoma, cervical cancer, kidney cancer, prostatecancer, lung cancer, synovial carcinoma pancreatic cancer, a sarcoma,glioma, head and neck cancer, gastric cancer, liver cancer, and othercarcinomas.
 69. The method of claim 68, characterized in that the saidcells are in frozen or fixed tissue or cells from said patient.
 70. Apolynucleotide encoding a polypeptide selected from the group consistingof SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 37, 38, 40, 42, 43, 45, 47,48, 49, 50, 52, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 74, 76,78 and
 80. 71. A polynucleotide according to claim 70 characterized inthat said polynucleotide has a sequence sharing at least 80% homologywith a polynucleotide selected from the group consisting of SEQ ID NOs:19, 21, 23, 25, 27, 29, 31, 33, 35, 39, 41, 44, 46, 51, 73, 75, 77, and79.
 72. A recombinant vector comprising the polynucleotide of claim 70.73. A host cell comprising the vector of claim
 72. 74. The host cell ofclaim 78, characterised in that it is selected from the group consistingof the hybridoma cell line designated 37.3D7 wherein said hybridoma cellline is deposited at the American Type Culture Collection under theaccession number PTA-7660; the hybridoma cell line designated 37.1F5,wherein said hybridoma cell line is deposited at the American TypeCulture Collection under the accession number PTA-7661; the hybridomacell line designated 53.2H11, wherein said hybridoma cell line isdeposited at the American Type Culture Collection under the accessionnumber PTA-7662; the hybridoma cell line designated EphA2-N1, whereinsaid hybridoma cell line is deposited at the American Type CultureCollection under the accession number PTM-8407; or the hybridoma cellline designated EphA2-N2, wherein said hybridoma cell line is depositedat the American Type Culture Collection under the accession numberPTM-8408.
 75. A pharmaceutical composition containing a conjugateaccording to claim 53 and a pharmaceutically acceptable carrier orexcipients.
 76. A conjugate according to claim 53 for use as amedicament.
 77. The use of a conjugate according to claim 53 to make amedicament to treat cancer.
 78. A host cell expressing the antibody ofclaim 1.